Method for treating cancer with c-kit antibodies

ABSTRACT

Disclosed herein are antibody molecules binding specifically to C-KIT, antigen-binding portions thereof and medical uses therefor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/521,793, filed on Jul. 25, 2019 and issued as U.S. Pat. No.10,611,838, which is a continuation of International Patent ApplicationNo. PCT/EP2019/053331, filed on Feb. 11, 2019, which claims the benefitof GB Patent Application No. 1806468.3, filed on Apr. 20, 2018, and GBPatent Application No. 1802201.2, filed on Feb. 9, 2018, the disclosureof each of which is hereby incorporated by reference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:UHFL_001_03US_SeqList_ST25.txt, date recorded: Mar. 13, 2020, file size˜101,357 bytes).

FIELD OF THE INVENTION

The invention relates to antibody molecules binding specifically toC-KIT (also known as KIT, Cluster of Differentiation 117 (CD117), PBT,SCFR, KIT proto-oncogene receptor tyrosine kinase) and medical usestherefor.

BACKGROUND OF THE INVENTION

C-KIT (also known as KIT, Cluster of Differentiation 117 (CD117), PBT,SCFR, KIT proto-oncogene receptor tyrosine kinase) is a transmembraneprotein that belongs to the immunoglobulin superfamily and binds to thesoluble factor SCF (stem cell factor). C-KIT is a receptor tyrosinekinase type III that is highly expressed by hematopoietic stem cells aswell as multiple other cell types, such as mature Mast Cells, where SCFsignalling acts as a cytokine. On binding to SCF, this receptordimerises, activating its tyrosine kinase activity. This kinaseactivation leads to further downstream activation of signal transductionmolecules that play known roles in cell survival, proliferation, anddifferentiation.

Altered forms of C-KIT, such as constitutively active mutants, arestrongly associated with the progression of several important types ofcancer, such as Gastrointestinal Stromal Tumours (GIST), Acute MyeloidLeukaemia (AML), Mast Cell tumours and Melanoma. Preclinical andclinical evidence suggests that blocking C-KIT-SCF signalling can haveclear therapeutic benefit in multiple cancers, but this haspredominantly been achieved using small molecule inhibitors of C-KITkinase function. Resistance mutations commonly develop after treatment,causing the therapeutic efficacy of the small molecule kinase inhibitorto be lost. Therapeutic antibodies that antagonise KIT signalling byblocking the ability of the receptors to dimerise have the potential toovercome kinase inhibitor resistance and mediate anti-tumour effects,via two mechanisms: 1. Potent inhibition of the KIT signalling pathwayby locking the receptors into a non-activating monomeric form. 2.Antibody effector-function mediated engagement of immune cells.Importantly, it has recently been recognised in preclinical studies thatC-KIT-SCF signalling in mast cells found in the tumour microenvironment(via SCF produced by stromal cells), can promote downstream cytokinesignalling that recruits myeloid cells such as Myeloid-DerivedSuppressor Cells (MDSCs). As MDSCs are believed to be a key cellpopulation that suppress immune responses against tumours, the indirectinhibition of their tumour infiltration via KIT signalling antagonismmay be an attractive therapeutic strategy.

The majority of currently approved antibody therapeutics are derivedfrom immunized rodents. Many of those antibodies have undergone aprocess known as “humanization”, via the “grafting” of murineComplementarity-Determining Regions (CDRs) into human v-gene frameworksequences (see Nelson et al., 2010, Nat Rev Drug Discov 9: 767-774).This process is often inaccurate and leads to a reduction in targetbinding affinity of the resulting antibody. To return the bindingaffinity of the original antibody, murine residues are usuallyintroduced at key positions in the variable domain frameworks of thegrafted v-domains (also known as “back-mutations”).

While antibodies humanized via CDR grafting and back mutations have beenshown to induce lower immune response rates in the clinic in comparisonto those with fully murine v-domains, antibodies humanized using thisbasic grafting method still carry significant clinical development risksdue to the potential physical instability and immunogenicity motifsstill housed in the grafted CDR loops. As animal testing of proteinimmunogenicity is often non-predictive of immune responses in man,antibody engineering for therapeutic use focuses on minimizing predictedhuman T-cell epitope content, non-human germline amino acid content andaggregation potential in the purified protein.

The ideal humanized antagonistic anti-C-KIT antibody would thereforehave as many residues as possible in the v-domains that are identical tothose found in both the frameworks and CDRs of well-characterized humangermline sequences. This high level of identity to high-stabilitygermlines that are highly expressed in the maximum number of potentialpatients minimises the risk of a therapeutic antibody having unwantedimmunogenicity in the clinic, or unusually high ‘cost of goods’ inmanufacturing.

Townsend et al. (2015; PNAS 112: 15354-15359) describe a method forgenerating antibodies in which CDRs derived from rat, rabbit and mouseantibodies were grafted into preferred human frameworks and then subjectto a human germ-lining approach termed “Augmented Binary Substitution”.Although the approach demonstrated a fundamental plasticity in theoriginal antibody paratopes, in the absence of highly accurateantibody-antigen co-crystal structural data, it is still not possible toreliably predict which individual residues in the CDR loops of any givenantibody can be converted to human germline, and in what combination.Additionally, the Townsend et al. study did not address the addition ofmutagenesis beyond the residues found in the human germline at positionswhere the removal of development risk motifs might be beneficial. Thisis a technological limitation which renders the process inherentlyunsatisfactory as it allows the retention of development liabilitymotifs in CDRs.

CDR germ-lining is thus a complex, multifactorial problem, as multiplefunctional properties of the molecule should preferably be maintained,including in this instance: target binding specificity, affinity toC-KIT from both human and animal test species (e.g. cynomolgus monkey,also known as the crab-eating macaque, i.e. Macaca fascicularis),v-domain biophysical stability and/or IgG yield from protein expressionplatforms used in research, clinical and commercial supply. Antibodyengineering studies have shown that mutation of even single residuepositions in key CDRs can have dramatic effects on all of these desiredmolecular properties.

WO2014018625A1 describes an antagonistic murine anti-C-KIT IgG moleculetermed “37M”, and also the preparation of humanized forms of 37M. Thosehumanized forms of 37M were produced using classical humanizationtechniques, i.e. by grafting of Kabat-defined murine CDRs into humanheavy and light chain framework sequences, with some of the humanframework residues being potentially back-mutated to the correspondinglypositioned 37M murine residues. For reasons noted above, such humanizedforms of 37M described in WO2014018625A1 are not ideal.

The present invention provides a number of optimized anti-C-KITantibodies and medical uses thereof.

SUMMARY OF THE INVENTION

According to one aspect of the invention, there is provided an antibodymolecule which specifically binds to human C-KIT, and optionally also tocynomolgus monkey C-KIT, or an antigen-binding portion thereof, whereinthe antibody molecule or antigen-binding portion comprises a heavy chainvariable region with:

an HCDR1 having amino acids in sequence in the following order:G-Y-T-F-T-D or a conservative substitution of D-Y or a conservativesubstitution of Y-Y-M or a conservative substitution of M-N(SEQ ID NO:1);

an HCDR2 having amino acids in sequence in the following order: M or aconservative substitution of M (for example, I)-G or a conservativesubstitution of G (for example, A)-R-I-Y-P-G or a conservativesubstitution of G (for example, A)-S or a conservative substitution of S(for example, A or T)-G-N-T-Y—Y-A-Q-K-F-Q-G (SEQ ID NO: 2); and

an HCDR3 having amino acids in sequence in the following order: G-V-Y orany amino acid (for example, W or F)-Y or any amino acid (for example, Eor H)-F or any amino acid (for example, Y, Q or L)-D or any amino acid(for example, G, N or S)-Y or any amino acid (for example, A, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, V) (SEQ ID NO: 3).

In aspects of the invention, the HCDR1 of the antibody molecule orantigen-binding portion may exclude the sequence GYTFTDYYIN (SEQ ID NO:4; 37M murine/humanized antibody HCDR1 disclosed in WO2014018625A1), theHCDR2 of the antibody molecule or antigen-binding portion may excludethe sequence IARIYPGSGNTYYNEKFKG (SEQ ID NO: 5; 37M murine/humanizedantibody HCDR2 disclosed in WO2014018625A1), and/or the HCDR3 of theantibody molecule or antigen-binding portion may exclude the sequenceGVYYFDY (SEQ ID NO: 6; 37M murine/humanized antibody HCDR3 disclosed inWO2014018625A1).

The antibody molecule or antigen-binding portion may further comprise alight chain variable region with:

an LCDR1 having amino acids in sequence in the following order:R-A-S-Q-G-I or a conservative substitution of I-R or a conservativesubstitution of R-T or any amino acid (such as N)-N or any amino acid(for example, Y)-L-A (SEQ ID NO: 7);

an LCDR2 having amino acids in sequence in the following order: A or anyamino acid (for example, S, Y)-A-S-S or any amino acid (for example,Y)-L or any amino acid (for example, R)-Q or any amino acid (forexample, Y)—S(SEQ ID NO: 8); and

an LCDR3 having amino acids in sequence in the following order: Q-Q-Y-Nor any amino acid (for example, A)-S or any amino acid (for example, Nor D)-Y-P-R-T (SEQ ID NO: 9).

In aspects of the invention, the LCDR1 of the antibody molecule orantigen-binding portion may exclude the sequence KASQNVRTNVA (SEQ ID NO:10; 37M murine/humanized antibody LCDR1 disclosed in WO2014018625A1),and/or the LCDR2 of the antibody molecule or antigen-binding portion mayexclude the sequence SASYRYS (SEQ ID NO: 11; 37M murine/humanizedantibody LCDR2 disclosed in WO2014018625A1). In some embodiments, theLCDR1 of the antibody molecule or antigen-binding portion may excludethe sequence KASQNVRTNVA (SEQ ID NO: 10; 37M murine/humanized antibodyLCDR1 disclosed in WO2014018625A1), and/or the LCDR2 of the antibodymolecule or antigen-binding portion may exclude the sequence SASYRYS(SEQ ID NO: 11; 37M murine/humanized antibody LCDR2 disclosed inWO2014018625A1) and/or the LCDR3 of the antibody molecule orantigen-binding portion may exclude the sequence QQYNSYPRT (SEQ ID NO:12; 37M murine/humanized antibody LCDR3 disclosed in WO2014018625A1).

In some aspects, disclosed herein is an anti-C-KIT antibody or anantigen-binding portion thereof, wherein the antibody comprises a heavychain variable (VH) region and a light chain variable (VL) region,wherein

-   -   (a) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYYDY (SEQ ID NO: 15); and the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNLA (SEQ ID NO:        16), LCDR2 of AASSLQS (SEQ ID NO: 24) and LCDR3 of QQYNSYPRT        (SEQ ID NO: 12);    -   (b) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYYDY (SEQ ID NO: 15); and the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNLA (SEQ ID NO:        16), LCDR2 of AASSLQS (SEQ ID NO: 24) and LCDR3 of QQYANYPRT        (SEQ ID NO: 25);    -   (c) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYYDY (SEQ ID NO: 15); and the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNLA (SEQ ID NO:        16), LCDR2 of SASSLQS (SEQ ID NO: 17) and LCDR3 of QQYANYPRT        (SEQ ID NO: 25);    -   (d) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYYDY (SEQ ID NO: 15); and the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNLA (SEQ ID NO:        16), LCDR2 of SASSLQS (SEQ ID NO: 17) and LCDR3 of QQYNSYPRT        (SEQ ID NO: 12);    -   (e) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYLDY (SEQ ID NO: 18); and the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNVA (SEQ ID NO:        19), LCDR2 of SASYRQS (SEQ ID NO: 20) and LCDR3 of QQYNSYPRT        (SEQ ID NO: 12);    -   (f) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYFDE (SEQ ID NO: 21); and the VL region        amino acid sequence comprises LCDR1 of RASQGVRTNLA (SEQ ID NO:        22), LCDR2 of AASSRQS (SEQ ID NO: 23) and LCDR3 of QQYNSYPRT        (SEQ ID NO: 12); or    -   (g) the VH region amino acid sequence comprises HCDR1 of        GYTFTDFYMN (SEQ ID NO: 180), HCDR2 of MGRIYPASGNTYYAQKFQG (SEQ        ID NO: 26) and HCDR3 of GVWYYDY (SEQ ID NO: 27); and the VL        region amino acid sequence comprises LCDR1 of RASQGIRTNLA (SEQ        ID NO: 16), LCDR2 of AASSLQS (SEQ ID NO: 24) and LCDR3 of        QQYANYPRT (SEQ ID NO: 25).

In some aspects, disclosed herein is an anti-C-KIT antibody or anantigen-binding portion thereof, wherein the antibody comprises a heavychain variable (VH) region and a light chain variable (VL) region,wherein

-   -   the VH region amino acid sequence comprises:    -   (a) HCDR1 of SEQ ID NO: 13;    -   (b) HCDR2 of SEQ ID NO: 14; and    -   (c) HCDR3 of SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 21; and    -   the VL region amino acid sequence comprises:    -   (a′) LCDR1 of SEQ ID NO: 16, SEQ ID NO: 19 or SEQ ID NO: 22;    -   (b′) LCDR2 of SEQ ID NO: 24, SEQ ID NO: 17, SEQ ID NO: 20 or SEQ        ID NO: 23 and    -   (c′) LCDR3 of SEQ ID NO: 12 or SEQ ID NO: 25.

In some aspects, disclosed herein is an anti-C-KIT antibody or anantigen-binding portion thereof, wherein the antibody comprises a heavychain variable (VH) region and a light chain variable (VL) region,wherein

-   -   (a) the VH region amino acid sequence comprises SEQ ID NO:185        and the VL region amino acid sequence comprises SEQ ID NO:186;    -   (b) the VH region amino acid sequence comprises SEQ ID NO:187        and the VL region amino acid sequence comprises SEQ ID NO:188;    -   (c) the VH region amino acid sequence comprises SEQ ID NO:189        and the VL region amino acid sequence comprises SEQ ID NO:190;    -   (d) the VH region amino acid sequence comprises SEQ ID NO:191        and the VL region amino acid sequence comprises SEQ ID NO:192;    -   (e) the VH region amino acid sequence comprises SEQ ID NO:193        and the VL region amino acid sequence comprises SEQ ID NO:194;        or    -   (f) the VH region amino acid sequence comprises SEQ ID NO:195        and the VL region amino acid sequence comprises SEQ ID NO:196.

In some aspects, disclosed herein is an anti-C-KIT antibody or anantigen-binding portion thereof, wherein the antibody comprises a heavychain variable (VH) region and a light chain variable (VL) region,wherein

-   -   (a) the HCDR1 comprises the amino acid sequence        G-Y-T-F-T-X₁-X₂-Y-X₃-N, wherein X₁ is D or a conservative        substitution of D, X₂ is Y or a conservative substitution of Y        and X₃ is M or a conservative substitution of M (SEQ ID NO: 1);    -   (b) the HCDR2 comprises        X₁-X₂-R-I-Y-P-X₃-X₄-G-N-T-Y-Y-A-Q-K-F-Q-G, wherein X₁ is M or a        conservative substitution of M, X₂ is G or a conservative        substitution of G, X₃ is G or a conservative substitution of G        and X₄ is S or a conservative substitution of S (SEQ ID NO: 2);    -   (c) the HCDR3 comprises G-V-X₁-X₂-X₃-X₄-X₅, wherein X₁ is Y or        any other amino acid, X₂ is Y or any other amino acid, X₃ is F        or any other amino acid, X₄ is D or any other amino acid and X₅        is Y or any other amino acid (SEQ ID NO: 3);    -   (d) the LCDR1 comprises R-A-S-Q-G-X₁-X₂-X₃-X₄-L-A, wherein X₁ is        I or a conservative substitution of I, X₂ is R or a conservative        substitution of R, X₃ is T or any other amino acid and X₄ is N        or any other amino acid (SEQ ID NO: 7);    -   (e) the LCDR2 comprises X₁-A-S-X₂-X₃-X₄-S, wherein X₁ is A or        any other amino acid, X₂ is S or any other amino acid, X₃ is L        or any other amino acid and X₄ is Q or any other amino acid (SEQ        ID NO: 8); and    -   (f) the LCDR3 comprises Q-Q-Y-X₁-X₂-Y-P-R-T, wherein X₁ is N or        any other amino acid and X₂ is S or any other amino acid (SEQ ID        NO: 9).

Also provided according to the invention is an immunoconjugatecomprising the antibody molecule or antigen-binding portion thereof asdefined herein linked to a therapeutic agent.

In another aspect the invention provides nucleic acid molecule encodingthe antibody molecule or antigen-binding portion thereof as definedherein.

Further provided is a vector comprising the nucleic acid molecule of theinvention.

Also provided is a host cell comprising the nucleic acid molecule or thevector of the invention as defined herein.

In a further aspect there is provided a method of producing ananti-C-KIT antibody and/or an antigen-binding portion thereof,comprising culturing the host cell of the invention under conditionsthat result in expression and/or production of the antibody and/or theantigen-binding portion thereof, and isolating the antibody and/or theantigen-binding portion thereof from the host cell or culture.

In another aspect of the invention there is provided a pharmaceuticalcomposition comprising the antibody molecule or antigen-binding portionthereof of the invention as defined herein, or the nucleic acid moleculeof the invention as defined herein, or the vector of the invention asdefined herein.

Further provided is a method for enhancing an immune response in asubject, comprising administering an effective amount of the antibodymolecule or antigen-binding portion thereof of the invention as definedherein, or the immunoconjugate of the invention as defined herein, orthe nucleic acid molecule of the invention as defined herein, or thevector of the invention as defined herein, or the pharmaceuticalcomposition of the invention as defined herein.

In a further aspect there is provided a method for treating orpreventing cancer in a subject, comprising administering an effectiveamount of the antibody molecule or antigen-binding portion thereof ofthe invention as defined herein, or the immunoconjugate of the inventionas defined herein, or the nucleic acid molecule of the invention asdefined herein, or the vector of the invention as defined herein, or thepharmaceutical composition of the invention as defined herein.

The invention also provides an antibody molecule or antigen-bindingportion thereof of the invention as defined herein, or theimmunoconjugate of the invention as defined herein, or the nucleic acidmolecule of the invention as defined herein, or the vector of theinvention as defined herein, or the pharmaceutical composition of theinvention as defined herein, for use in the treatment of cancer.

In another aspect the invention provides the antibody molecule, orantigen-binding portion thereof, or the immunoconjugate, or the nucleicacid molecule, or the vector for use, or the method of treatment of theinvention as defined herein, for separate, sequential or simultaneoususe in a combination with a second therapeutic agent, for example ananti-cancer agent.

In a further aspect there is provided the use of an antibody molecule orantigen-binding portion thereof of the invention as defined herein, oran immunoconjugate of the invention as defined herein, or a nucleic acidmolecule of the invention as defined herein, or a vector of theinvention as defined herein, or a pharmaceutical composition of theinvention as defined herein, in the manufacture of a medicament for thetreatment of cancer.

The invention also provides a method for treating or preventing anautoimmune disease or an inflammatory disease in a subject, comprisingadministering an effective amount of the antibody molecule orantigen-binding portion thereof as defined herein, or theimmunoconjugate as defined here, or the nucleic acid molecule as definedherein, or the vector as defined herein, or the pharmaceuticalcomposition as defined herein.

The autoimmune disease or inflammatory disease may be selected in allaspects from the group consisting of: arthritis, asthma, multiplesclerosis, psoriasis, Crohn's disease, inflammatory bowel disease,lupus, Grave's disease and Hashimoto's thyroiditis, and ankylosingspondylitis.

Also provided is an antibody molecule or antigen-binding portion thereofas defined herein, or the immunoconjugate as defined herein, or thenucleic acid molecule as defined herein, or the vector as definedherein, or the pharmaceutical composition as defined herein, for use inthe treatment of an autoimmune disease or an inflammatory disease.

Further provided is the use of an antibody molecule or antigen-bindingportion thereof as defined herein, or an immunoconjugate as definedherein, or a nucleic acid molecule as defined herein, or a vector asdefined herein, or a pharmaceutical composition as defined herein, inthe manufacture of a medicament for the treatment of an autoimmunedisease or an inflammatory disease.

The invention also provides a method for treating or preventing acardiovascular disease or a fibrotic disease in a subject, comprisingadministering an effective amount of the antibody molecule orantigen-binding portion thereof as defined herein, or theimmunoconjugate as defined here, or the nucleic acid molecule as definedherein, or the vector as defined herein, or the pharmaceuticalcomposition as defined herein.

Further provided herein is an antibody molecule or antigen-bindingportion thereof as defined herein, or the immunoconjugate as definedherein, or the nucleic acid molecule as defined herein, or the vector asdefined herein, or the pharmaceutical composition as defined herein, foruse as a medicament. Also provided is an antibody molecule orantigen-binding portion thereof as defined herein, or theimmunoconjugate as defined herein, or the nucleic acid molecule asdefined herein, or the vector as defined herein, or the pharmaceuticalcomposition as defined herein, for use in the treatment of acardiovascular disease or a fibrotic disease.

Further provided is the use of an antibody molecule or antigen-bindingportion thereof as defined herein, or an immunoconjugate as definedherein, or a nucleic acid molecule as defined herein, or a vector asdefined herein, or a pharmaceutical composition as defined herein, inthe manufacture of a medicament for the treatment of an autoimmunedisease, an inflammatory disease or a fibrotic disease.

The cardiovascular disease in any aspect of the invention may forexample be coronary heart disease or atherosclerosis.

The fibrotic disease in any aspect of the invention may be selected fromthe group consisting of myocardial infarction, angina, osteoarthritis,pulmonary fibrosis, cystic fibrosis, bronchitis and asthma.

The invention also provides a method of producing an antibody moleculewhich specifically binds to human C-KIT and optionally also tocynomolgus monkey C-KIT, or an antigen-binding portion thereof,comprising the steps of:

(1) grafting anti-C-KIT CDRs from a non-human source into a humanv-domain framework to produce a humanized anti-C-KIT antibody moleculeor antigen-binding portion thereof;

(2) generating a phage library of clones of the humanized anti-C-KITantibody molecule or antigen-binding portion thereof comprising one ormore mutations in the CDRs;

(3) screening the phage library for binding to human C-KIT andoptionally also to cynomolgus monkey C-KIT;

(4) selecting clones from the screening step (3) having bindingspecificity to human C-KIT and optionally also to cynomolgus monkeyC-KIT; and

(5) producing an antibody molecule which specifically binds to humanC-KIT and optionally also to cynomolgus monkey C-KIT, or anantigen-binding portion thereof from clones selected from step (4).

The method may comprise a further step of producing additional clonesbased on the clones selected in step (4), for example based on furtherexploratory mutagenesis at specific positions in the CDRs of the clonesselected in step (4), to enhance humanization and/or minimise human Tcell epitope content and/or improve manufacturing properties in theantibody molecule or antigen-binding portion thereof produced in step(5).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1B. Direct binding ELISA and HTRF competition screening oflibrary-derived anti-C-KIT scFvs against human and cyno C-KIT-Fcproteins. Clones were derived from separate phage selection branches onbiotinylated human and/or cynomolgus monkey C-KIT-Fc proteins in eachround. After multiple rounds of selection, library-derived clones (blackcircles) were screened for binding against both human (FIG. 1A) and cyno(FIG. 1B) C-KIT-Fc and epitope blocking of h37M IgG1 against bothorthologs. Negative control (non-C-KIT-binding) and positive controlh37M scFvs are represented in grey triangles and squares, respectively.

FIG. 2A-FIG. 2B. Analysis of CDR residue tolerance for mutation togermline. A plot of murine amino acid retention frequencies in the CDRs(SEQ ID NOs: 4, 5 and 6) of the ELISA-positive population of 219 uniquescFv clones is shown for V_(H) (FIG. 2A) and V_(L) (FIG. 2B) domains,respectively. Only those residues targeted for human/murine residuemutagenesis are plotted, other than in the HCDR3. CDR residues noted inparentheses on the X-axes were identical to those found in the humangermlines used for grafting (IGKV1-16 and IGHV1-46). Those residues inthe HCDR2 that are not in parentheses, but whose values are set at 0,were mutated to human germline during the grafting process. In bothplots the dashed line in grey at 75% represents the cut off fortolerance of murine residue replacement by human germline.

FIG. 3A-FIG. 3B. Direct titration ELISA for IgG binding to human andcyno C-KIT-Fc proteins. Chimeric anti-C-KIT (m37M), humanized h37M,library-derived and designer clones in human IgG1 format were titrated(in μg/ml) in a direct binding ELISA against human (FIG. 3A) and cyno(FIG. 3B) C-KIT-Fc proteins. The 37M, library-derived clones anddesigner clone MH demonstrated binding activity against both orthologsof C-KIT. All library derived and several designer clones retainedapproximately equivalent or improved human and cyno C-KIT binding, whileall TTP clones and MH clones 4, 6, 7, 9, 13, 14 and 15 all exhibitedreduced binding signal on one or both orthologs.

FIG. 4A-FIG. 4E. HTRF-based solution-phase, high-sensitivity, C-KITepitope competition assay. HTRF binding signal for the h37M IgG to humanor cyno C-KIT was examined in the presence of titrated competitor IgGsincluding library-derived and designer leads, plus Isotype IgG1 as anegative control and unlabelled h37M IgG1 as a positive control. Alllibrary-derived and multiple designer IgGs exhibited fullconcentration-dependent inhibition of h37M binding to human (FIG. 4A)and cyno (FIG. 4C, FIG. 4D) c-KIT, similar to unlabelled h37M IgG,suggesting maintenance of a shared epitope and binding affinity. All TTPclones, plus MH clones 4, 6, 7 and 9 were found to have reduced abilityto inhibit h37M binding to either human (FIG. 4B) or cyno (FIG. 4E)C-KIT.

FIG. 5A-FIG. 5F. Flow cytometric binding to human and cyno C-KIT+ CHO-K1cells for library-derived and primary designer leads. Anti-C-KITcontrols m37M and h37M, library-derived and designer leads in IgG1format were examined for specific binding on human C-KIT-transfectedCHO-K1 cells (FIG. 5A, FIG. 5B), cyno C-KIT-transfected CHO-K1 cells(FIG. 5C, FIG. 5D), and wild type (WT, i.e. untransfected) CHO-K1 cells(FIG. 5E, FIG. 5F). IgGs were tested at concentrations ranging from0.02-100 μg/ml. Concentration-dependent binding was observed againstboth human and cyno cell lines for all C-KIT-specific antibodies but notisotype controls. No binding signals above background were observedagainst wild type CHO-K1 cells.

FIG. 6A-FIG. 6B. Direct titration ELISA for second-generation designerIgGs binding to human and cyno C-KIT-Fc proteins. Anti-C-KIT h37M, MH5and second-generation designer clones in human IgG1 format were titrated(in μg/ml) in a direct binding ELISA against human (FIG. 6A) and cyno(FIG. 6B) C-KIT-Fc proteins. The h37M and MH5 clones demonstratedbinding activity against both orthologs of C-KIT. All designer clonesother than MH5.33 retained human and cyno C-KIT binding, but only clonesMH5.1, 5.2, 5.3, 5.22, 5.23, 5.24, 5.34 and 5.35 exhibited bindingcomparable to clone h37M on both orthologs.

FIG. 7A-FIG. 7B. HTRF-based C-KIT epitope competition assay for keysecond-generation designer leads. HTRF binding signal for the h37M IgGto human or cyno C-KIT was examined in the presence of titratedcompetitor second-generation designer leads in IgG1 format, plus isotypeIgG1 as a negative control and unlabelled h37M IgG1 as a positivecontrol. All IgGs (other than the Isotype control IgG1) exhibited fullconcentration-dependent inhibition of h37M binding to human (FIG. 7A)and cyno (FIG. 7B) C-KIT, similar to unlabelled h37M IgG.

FIG. 8A-FIG. 8C. Flow cytometric binding to human and cyno C-KIT+ CHO-K1cells for second-generation designer leads. Anti-C-KIT library-derivedand designer leads in IgG1 format were examined for specific binding onhuman C-KIT-transfected CHO-K1 cells (FIG. 8A), cyno C-KIT-transfectedCHO-K1 cells (FIG. 8B), and wild type (wt, i.e. untransfected) CHO-K1cells (FIG. 8C). IgGs were tested at concentrations ranging from24-100,000 ng/ml. Concentration-dependent binding was observed againstboth human and cyno cell lines for all C-KIT-specific antibodies but notIsotype controls. No binding signals above background were observedagainst wild type CHO-K1 cells.

FIG. 9A-FIG. 9F. Binding specificity analyses for prioritized leadclones. Off-target homologue binding risk for h37M and multiple leadantibodies in IgG1 format at 1 μg/ml was examined by direct ELISA onC-KIT-Fc orthologs and a panel of 14 human immunoglobulin superfamilyproteins. For all IgGs, binding was observed to human and cyno C-KIT-Fcalone. No binding above background was observed for any other human orortholog protein.

FIG. 10A-FIG. 10C. Development risk ELISAs. This assay showed that theE-C7, E-C3, MH1, MH5, MH5.22 and h37M antibodies in IgG1 form exhibitequivalent or lower binding to the negatively charged biomoleculesInsulin (FIG. 10A), double-stranded DNA (dsDNA) (FIG. 10B) andsingle-stranded DNA (ssDNA) (FIG. 10C) than the negative control IgG1Ustekinumab analogue. Strong off-target binding to these molecules, asobserved for Bococizumab and Briakinumab analogues has been shown to bea high-risk indicator of poor pharmacokinetics of therapeuticantibodies.

FIG. 11A-FIG. 11H. T cell epitope peptide content in lead antibodyv-domains. The v-domains of h37M (FIG. 11A), E-C7 (FIG. 11B), F-C5 (FIG.11C), E-C3 (FIG. 11D), MH1 (FIG. 11E), MH5 (FIG. 11F), MH5.22 (FIG. 11G)and MH5-DI (FIG. 11H) antibodies were examined for the presence ofGermline (GE), High Affinity Foreign (HAF), Low Affinity Foreign (LAF)and TCED+ T cell receptor epitopes. Both the VH and VL domains of h37Mwere found to contain multiple high-risk human T cell epitopes and fewgermline epitopes. In all lead clones, the high-risk epitope content wassignificantly reduced and germline epitope content improved.

FIG. 12A-FIG. 12C. Affinity analyses for IgG1 Fc engineered variants onhuman FcγRIIb. Purified anti-KIT IgGs were analysed for binding affinityto FcγRIIb in a ‘steady state’ affinity measurement on a BIACORE® T200instrument. Clones h37M-IgG1 (FIG. 12A), h37M-IgG1-3M (FIG. 12B) andMH1-IgG-4M (FIG. 12C) all exhibited measurable binding to FcγRIIb, withMH1-IgG-4M showing the lowest overall binding signal across the fullconcentration range. In A-C, the raw data for chip interaction is shownin each upper panel and the plotted curves for Response Units (RU) perconcentration are shown in the lower panel.

FIG. 13A-FIG. 13C. Human proteome re-array analyses. After a full screenof 5528 unique proteins, analyses of binding specificity were performedon chips in which plasmids encoding potentially relevant targets werearrayed and used to transfect HEK293 cells. Transfection of all plasmidswas confirmed by screening for the co-encoded marker ZS green (FIG.13A). Separate chips were then probed using Rituximab analog andsecondary labelled antibody only (FIG. 13A), h37M at 0.5 and 2 μg/ml(FIG. 13B) and MH1 at 0.5 and 2 μg/ml (FIG. 13C). These analysesconfirmed that h37M and MH1 both exhibited highly specific binding toC-KIT. Binding signals to MMP7, CRIM1 and F13A1 on all chips were foundto be an artefact of the secondary antibody (FIG. 13A).

FIG. 14A-FIG. 14C. Anti-C-KIT cell killing assays. Internalisation andtoxin delivery was examined on CHO cells transfected with human (FIG.14A) and cyno (FIG. 14B) C-KIT, and the human erythroleukaemia cell lineTF-1 (FIG. 14C), in the presence of FabZAP reagent. Clones h37M, MH1 andMH5-DI in IgG1-3M format all drove highly similar, high-potency cellkilling.

FIG. 15A-FIG. 15B. HTRF-based C-KIT epitope competition assay for h37M,MH1, MH5. HTRF binding signal for the h37M IgG to human or cyno C-KITwas examined in the presence of titrated competitor MH1 and MH5 inIgG1-3M format, plus isotype IgG1 as a negative control and unlabelledh37M IgG1-3M as a positive control. All IgGs (other than the Isotypecontrol IgG1) exhibited full concentration-dependent inhibition of h37Mbinding to human (FIG. 15A) and cyno (FIG. 15B) C-KIT, overlapping withunlabelled h37M.

FIG. 16. TF-1 cell-based assay for inhibition of C-KIT/SCF-drivencellular proliferation. Human TF-1 cell line was cultured in thepresence of SCF, which promotes cell proliferation through C-KIT.Antibodies were applied for 72 hours and proliferation measured viaresazurin fluorescence. h37M was found to be unexpectedly significantlymore potent than any other antibody tested.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention, there is provided anantibody molecule which specifically binds to human C-KIT and optionallyalso to cynomolgus monkey C-KIT, or an antigen-binding portion thereof,wherein the antibody molecule or antigen-binding portion comprises aheavy chain variable region with:

an HCDR1 (heavy chain complementarity determining region 1) having aminoacids in sequence in the following order: G-Y-T-F-T-D or a conservativesubstitution of D-Y or a conservative substitution of Y-Y-M or aconservative substitution of M-N(SEQ ID NO: 1);

an HCDR2 (heavy chain complementarity determining region 2) having aminoacids in sequence in the following order: M or a conservativesubstitution of M (for example, I)-G or a conservative substitution of G(for example, A)-R-I-Y-P-G or a conservative substitution of G (forexample, A)-S or a conservative substitution of S (for example, A orT)-G-N-T-Y-Y-A-Q-K-F-Q-G (SEQ ID NO: 2); and

an HCDR3 (heavy chain complementarity determining region 3) having aminoacids in sequence in the following order: G-V-Y or any amino acid (forexample, W or F)-Y or any amino acid (for example, E or H)-F or anyamino acid (for example, Y, Q or L)-D or any amino acid (for example, G,N or S)-Y or any amino acid (for example, A, D, E, F, G, H, I, K, L, M,N, P, Q, R, S, T, V) (SEQ ID NO: 3).

In some aspects an anti-C-KIT antibody or antigen-binding portionprovided herein specifically binds to a C-KIT protein comprising orconsisting of SEQ ID NO:208. In some aspects an anti-C-KIT antibody orantigen-binding portion provided herein specifically binds to a C-KITprotein having an amino acid sequence that is at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98% or at least about 99% identical to SEQ ID NO:208. Insome aspects an anti-C-KIT antibody or antigen-binding portion providedherein specifically binds to a C-KIT protein encoded by a nucleic acidmolecule comprising or consisting of SEQ ID NO:209 or SEQ ID NO:210.

In aspects of the invention, the HCDR1 of the antibody molecule orantigen-binding portion may exclude the sequence GYTFTDYYIN (SEQ ID NO:4; 37M murine/humanized antibody HCDR1 disclosed in WO2014018625A1), theHCDR2 of the antibody molecule or antigen-binding portion may excludethe sequence IARIYPGSGNTYYNEKFKG (SEQ ID NO: 5; 37M murine/humanizedantibody HCDR2 disclosed in WO2014018625A1), and/or the HCDR3 of theantibody molecule or antigen-binding portion may exclude the sequenceGVYYFDY (SEQ ID NO: 6; 37M murine/humanized antibody HCDR3 disclosed inWO2014018625A1).

The antibody molecule or antigen-binding portion may further comprise alight chain variable region with:

an LCDR1 (light chain complementarity determining region 1) having aminoacids in sequence in the following order: R-A-S-Q-G-I or a conservativesubstitution of I-R or a conservative substitution of R-T or any aminoacid (such as N)-N or any amino acid (for example, Y)-L-A (SEQ ID NO:7);

an LCDR2 (light chain complementarity determining region 2) having aminoacids in sequence in the following order: A or any amino acid (forexample, S, Y)-A-S-S or any amino acid (for example, Y)-L or any aminoacid (for example, R)-Q or any amino acid (for example, Y)-S(SEQ ID NO:8); and

an LCDR3 (light chain complementarity determining region 3) having aminoacids in sequence in the following order: Q-Q-Y-N or any amino acid (forexample, A)-S or any amino acid (for example, N or D)-Y-P-R-T (SEQ IDNO: 9).

In some aspects of the invention, the LCDR1 of the antibody molecule orantigen-binding portion may exclude the sequence KASQNVRTNVA (SEQ ID NO:10; 37M murine/humanized antibody LCDR1 disclosed in WO2014018625A1),and/or the LCDR2 of the antibody molecule or antigen-binding portion mayexclude the sequence SASYRYS (SEQ ID NO: 11; 37M murine/humanizedantibody LCDR2 disclosed in WO2014018625A1). In some embodiments, theLCDR1 of the antibody molecule or antigen-binding portion may excludethe sequence KASQNVRTNVA (SEQ ID NO: 10; 37M murine/humanized antibodyLCDR1 disclosed in WO2014018625A1), and/or the LCDR2 of the antibodymolecule or antigen-binding portion may exclude the sequence SASYRYS(SEQ ID NO: 11; 37M murine/humanized antibody LCDR2 disclosed inWO2014018625A1) and/or the LCDR3 of the antibody molecule orantigen-binding portion may exclude the sequence QQYNSYPRT (SEQ ID NO:12; 37M murine/humanized antibody LCDR3 disclosed in WO2014018625A1).

In some aspects, disclosed herein is an anti-C-KIT antibody or anantigen-binding portion thereof, wherein the antibody comprises a heavychain variable (VH) region and a light chain variable (VL) region,wherein

-   -   (a) the HCDR1 comprises the amino acid sequence        G-Y-T-F-T-X₁-X₂-Y-X₃-N, wherein X₁ is D or a conservative        substitution of D, X₂ is Y or a conservative substitution of Y        and X₃ is M or a conservative substitution of M (SEQ ID NO: 1);    -   (b) the HCDR2 comprises        X₁-X₂-R-I-Y-P-X₃-X₄-G-N-T-Y-Y-A-Q-K-F-Q-G, wherein X₁ is M or a        conservative substitution of M (for example, I), X₂ is G or a        conservative substitution of G (for example, A), X₃ is G or a        conservative substitution of G (for example, A) and X₄ is S or a        conservative substitution of S (for example, A or T) (SEQ ID NO:        2);    -   (c) the HCDR3 comprises G-V-X₁-X₂-X₃-X₄-X₅, wherein X₁ is Y or        any other amino acid (for example, W or F), X₂ is Y or any other        amino acid (for example, E or H), X₃ is F or any other amino        acid (for example, Y, Q or L), X₄ is D or any other amino acid        (for example, G, N or S) and X₅ is Y or any other amino acid        (for example, A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T        or V) (SEQ ID NO: 3);    -   (d) the LCDR1 comprises R-A-S-Q-G-X₁-X₂-X₃-X₄-L-A, wherein X₁ is        I or a conservative substitution of I, X₂ is R or a conservative        substitution of R, X₃ is T or any other amino acid (for        example, N) and X₄ is N or any other amino acid (for example, Y)        (SEQ ID NO: 7);    -   (e) the LCDR2 comprises X₁-A-S-X₂-X₃-X₄-S, wherein X₁ is A or        any other amino acid (for example, S or Y), X₂ is S or any other        amino acid (for example, Y), X₃ is L or any other amino acid        (for example, R) and X₄ is Q or any other amino acid (for        example, Y) (SEQ ID NO: 8); and    -   (f) the LCDR3 comprises Q-Q-Y-X₁-X₂-Y-P-R-T, wherein X₁ is N or        any other amino acid (for example, A) and X₂ is S or any other        amino acid (for example, N or D) (SEQ ID NO: 9).

In some aspects, disclosed herein is an anti-C-KIT antibody or anantigen-binding portion thereof, wherein the antibody comprises a heavychain variable (VH) region comprising, in amino-terminal tocarboxyl-terminal order, FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4 and a lightchain variable (VL) region comprising, in amino-terminal tocarboxyl-terminal order, FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, wherein theHCDR1 is SEQ ID NO:1, the HCDR2 is SEQ ID NO:2, the HCDR3 is SEQ IDNO:3, the LCDR1 is SEQ ID NO:7, the LCDR2 is SEQ ID NO:8 and the LCDR3is SEQ ID NO:9, wherein the heavy chain FR1, FR2, FR3 and FR4 amino acidsequences are the heavy chain FR1, FR2, FR3 and FR4 amino acid sequencesin SEQ ID NO: 89 (see Table 2) and wherein the light chain FR1, FR2, FR3and FR4 amino acid sequences are the light chain FR1, FR2, FR3 and FR4amino acid sequences in SEQ ID NO: 92 (see Table 2).

As elaborated herein, the present inventors have succeeded for the firsttime in generating a number of optimized anti-C-KIT antibody moleculesusing CDR sequences derived from the murine anti-C-KIT antibody 37Mdisclosed in WO2014018625A1. In embodiments of the present invention,these antibody molecules have been selected to have binding specificityto both human C-KIT as well as cynomolgus monkey C-KIT (to facilitate invivo studies in an appropriate animal test species). Further refining ofthe optimized antibody molecules as described herein has providedimproved variable domain stability, higher expression yields, and/orreduced immunogenicity.

Preferred optimized anti-C-KIT antibody molecules of the presentinvention do not necessarily have the maximum number of human germlinesubstitutions at corresponding murine CDR or other (such as framework)amino acid positions. As elaborated in the experimental section below,we have found that “maximally humanized” antibody molecules are notnecessary “maximally optimized” in terms of anti-C-KIT bindingcharacteristics and/or other desirable features.

The present invention encompasses modifications to the amino acidsequence of the antibody molecule or antigen-binding portion thereof asdefined herein. For example, the invention includes antibody moleculesand corresponding antigen-binding portions thereof comprisingfunctionally equivalent variable regions and CDRs which do notsignificantly affect their properties as well as variants which haveenhanced or decreased activity and/or affinity. For example, the aminoacid sequence may be mutated to obtain an antibody with the desiredbinding affinity to C-KIT. Insertions which include amino- and/orcarboxyl-terminal fusions ranging in length from one residue topolypeptides containing a hundred or more residues, as well asintrasequence insertions of single or multiple amino acid residues, areenvisaged. Examples of terminal insertions include an antibody moleculewith an N-terminal methionyl residue or the antibody molecule fused toan epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the half-life of the antibody in theblood circulation.

The antibody molecule or antigen-binding portion of the invention mayinclude glycosylated and nonglycosylated polypeptides, as well aspolypeptides with other post-translational modifications, such as, forexample, glycosylation with different sugars, acetylation, andphosphorylation. The antibody molecule or antigen-binding portion of theinvention may be mutated to alter such post-translational modifications,for example by adding, removing or replacing one or more amino acidresidues to form or remove a glycosylation site.

The antibody molecule or antigen-binding portion of the invention may bemodified for example by amino acid substitution to remove potentialproteolytic sites in the antibody.

In the antibody molecule or antigen-binding portion thereof, the HCDR1may have the amino acid sequence: G-Y-T-F-T-D/N-Y/H/F-Y-M/I-N(SEQ ID NO:28); the HCDR2 may have the amino acid sequence:M/I-G/A-R-I-Y-P-G/A-S/T/A-G-N-T-Y-Y-A-Q-K-F-Q-G (SEQ ID NO: 29); and theHCDR3 may have the amino acid sequence:G-V-Y/W/F-Y/E/H-F/Y/Q/L-D/G/N/S-Y/NE/F/GH/I/K/L/M/N/P/Q/R/S/TN (SEQ IDNO: 30).

For example, the HCDR1 may have the amino acid sequence:G-Y-T-F-T-D/N-Y/F-Y-M-N (SEQ ID NO: 31); the HCDR2 may have the aminoacid sequence: M/I-G-R-I-Y-P-G/A-S-G-N-T-Y-Y-A-Q-K-F-Q-G (SEQ ID NO:32); and the HCDR3 may have the amino acid sequence:G-V-Y/W-Y/E/H-Y/Q/L-D-Y/S/T/E (SEQ ID NO: 33).

In the antibody molecule or antigen-binding portion thereof, the LCDR1may have the amino acid sequence: R-A-S-Q-G-IN-R-T/N-N-L-A (SEQ ID NO:34); the LCDR2 may have the amino acid sequence: A/S/Y-A-S-S-L-Q-S(SEQID NO: 35); and the LCDR3 may have the amino acid sequence:Q-Q-Y-N/A/S/E/T-S/A/N/D-Y-P-R-T (SEQ ID NO: 36).

For example, the LCDR1 may have the amino acid sequence:R-A-S-Q-G-I-R-T/N-N-L-A (SEQ ID NO: 37); the LCDR2 may have the aminoacid sequence: A-A-S-S-L-Q-S(SEQ ID NO: 24); and the LCDR3 may have theamino acid sequence: Q-Q-Y-N/A-S/N-Y-P-R-T (SEQ ID NO: 38).

In specific embodiments of the invention, the antibody molecule orantigen-binding portion may comprise:

(a) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVWYFDY (HCDR3; SEQ ID NO:40), RASQGVRTNVA (LCDR1; SEQ ID NO: 39), SASSLQS (LCDR2; SEQ ID NO: 17),QQYNSYPRT (LCDR3; SEQ ID NO: 12), [Clone E-E10]; or

(b) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYQDY (HCDR3; SEQ ID NO:41), RASQGVRTNVA (LCDR1; SEQ ID NO: 39), AASSRQS (LCDR2; SEQ ID NO: 23)and QQYNSYPRT (LCDR3; SEQ ID NO: 12) [Clone F-F2];

(c) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYEFDY (HCDR3; SEQ ID NO:42), RASQGVRNNLA (LCDR1; SEQ ID NO: 43), AASYRQS (LCDR2; SEQ ID NO: 44)and QQYNSYPRT (LCDR3; SEQ ID NO: 12) [Clone C-B12];

(d) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),IGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 45), GVYYFDS (HCDR3; SEQ ID NO:46), RASQGVRNNVA (LCDR1; SEQ ID NO: 47), YASSLQS (LCDR2; SEQ ID NO: 48)and QQYNSYPRT (LCDR3; SEQ ID NO: 12) [Clone C-A7];

(e) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYHFDY (HCDR3; SEQ ID NO:49), RASQGVRNNVA (LCDR1; SEQ ID NO: 47), AASYLQS (LCDR2; SEQ ID NO: 50)and QQYNSYPRT (LCDR3; SEQ ID NO: 12) [Clone C-A5];

(f) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYFDT (HCDR3; SEQ ID NO:51), RASQGVRTNLA (LCDR1; SEQ ID NO: 22), AASSRQS (LCDR2; SEQ ID NO: 23)and QQYNSYPRT (LCDR3; SEQ ID NO: 12) [Clone D-A10];

(g) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), SASSLQS (LCDR2; SEQ ID NO: 17)and QQYNSYPRT (LCDR3; SEQ ID NO: 12) [Clone E-C7];

(h) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGVRTNLA (LCDR1; SEQ ID NO: 22), SASSLQS (LCDR2; SEQ ID NO: 17)and QQYNSYPRT (LCDR3; SEQ ID NO: 12) [Clone D-D5];

(i) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYLDY (HCDR3; SEQ ID NO:18), RASQGIRTNVA (LCDR1; SEQ ID NO: 19), SASYRQS (LCDR2; SEQ ID NO: 20)and QQYNSYPRT (LCDR3; SEQ ID NO: 12) [Clone E-C2];

(j) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYLDY (HCDR3; SEQ ID NO:18), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), AASYRQS (LCDR2; SEQ ID NO: 44)and QQYNSYPRT (LCDR3; SEQ ID NO: 12) [Clone F-B11];

(k) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYLDY (HCDR3; SEQ ID NO:18), RASQGVRTNVA (LCDR1; SEQ ID NO: 39), SASSLQS (LCDR2; SEQ ID NO: 17)and QQYNSYPRT (LCDR3; SEQ ID NO: 12) [Clone D-D9];

(l) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYFDE (HCDR3; SEQ ID NO:21), RASQGVRTNVA (LCDR1; SEQ ID NO: 39), SASSRQS (LCDR2; SEQ ID NO: 52)and QQYNSYPRT (LCDR3; SEQ ID NO: 12) [Clone E-G7];

(m) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYFDE (HCDR3; SEQ ID NO:21), RASQGVRTNLA (LCDR1; SEQ ID NO: 22), AASSRQS (LCDR2; SEQ ID NO: 23)and QQYNSYPRT (LCDR3; SEQ ID NO: 12) [Clone F-05];

(n) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), AASSLQS (LCDR2; SEQ ID NO: 24)and QQYNSYPRT (LCDR3; SEQ ID NO: 12) [Clone MH1];

(o) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), SASSLQS (LCDR2; SEQ ID NO: 17)and QQYASYPRT (LCDR3; SEQ ID NO: 53) [Clone MH2];

(p) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), SASSLQS (LCDR2; SEQ ID NO: 17)and QQYNAYPRT (LCDR3; SEQ ID NO: 54) [Clone MH3];

(q) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), SASSLQS (LCDR2; SEQ ID NO: 17)and QQYNDYPRT (LCDR3; SEQ ID NO: 55) [Clone MH4];

(r) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), SASSLQS (LCDR2; SEQ ID NO: 17)and QQYANYPRT (LCDR3; SEQ ID NO: 25) [Clone MH5];

(s) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), SASSLQS (LCDR2; SEQ ID NO: 17)and QQYNSYPKT (LCDR3; SEQ ID NO: 56) [Clone MH6];

(t) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), SASSLQS (LCDR2; SEQ ID NO: 17)and QQYNSYPHT (LCDR3; SEQ ID NO: 57) [Clone MH7];

(u) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), SASSLQS (LCDR2; SEQ ID NO: 17)and QQYSSYPRT (LCDR3; SEQ ID NO: 58) [Clone MH8];

(v) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), SASSLQS (LCDR2; SEQ ID NO: 17)and QQYESYPRT (LCDR3; SEQ ID NO: 59) [Clone MH9];

(w) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), SASSLQS (LCDR2; SEQ ID NO: 17)and QQYTSYPRT (LCDR3; SEQ ID NO: 60) [Clone MH10];

(x) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), AASSLQS (LCDR2; SEQ ID NO: 24)and QQYNAYPRT (LCDR3; SEQ ID NO: 54) [Clone MH11];

(y) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGVRTNLA (LCDR1; SEQ ID NO: 22), AASSLQS (LCDR2; SEQ ID NO: 24)and QQYNSYPRT (LCDR3; SEQ ID NO: 12) [Clone MH12];

(z) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGVRTNLA (LCDR1; SEQ ID NO: 22), AASSLQS (LCDR2; SEQ ID NO: 24)and QQYNAYPRT (LCDR3; SEQ ID NO: 54) [Clone MH13];

(aa) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGVRTNLA (LCDR1; SEQ ID NO: 22), AASSLQS (LCDR2; SEQ ID NO: 24)and QQYNSYPRT (LCDR3; SEQ ID NO: 12) [Clone MH14];

(bb) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGVRTNLA (LCDR1; SEQ ID NO: 22), AASSLQS (LCDR2; SEQ ID NO: 24)and QQYNAYPRT (LCDR3; SEQ ID NO: 54) [Clone MH15];

(cc) the amino acid sequences GYTFTNYYMN (HCDR1; SEQ ID NO: 181),MGRIYPGTGNTYYAQKFQG (HCDR2; SEQ ID NO: 61), GVWYYDY (HCDR3; SEQ ID NO:27), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), AASSLQS (LCDR2; SEQ ID NO: 24)and QQYANYPRT (LCDR3; SEQ ID NO: 25) [Clone MH5.1];

(dd) the amino acid sequences GYTFTDFYMN (HCDR1; SEQ ID NO: 180),MGRIYPGTGNTYYAQKFQG (HCDR2; SEQ ID NO: 61), GVWYYDY (HCDR3; SEQ ID NO:27), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), AASSLQS (LCDR2; SEQ ID NO: 24)and QQYANYPRT (LCDR3; SEQ ID NO: 25) [Clone MH5.2];

(ee) the amino acid sequences GYTFTDFYMN (HCDR1; SEQ ID NO: 180),MGRIYPASGNTYYAQKFQG (HCDR2; SEQ ID NO: 26), GVWYYDY (HCDR3; SEQ ID NO:27), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), AASSLQS (LCDR2; SEQ ID NO: 24)and QQYANYPRT (LCDR3; SEQ ID NO: 25) [Clone MH5.22];

(ff) the amino acid sequences GYTFTDFYMN (HCDR1; SEQ ID NO: 180),MGRIYPASGNTYYAQKFQG (HCDR2; SEQ ID NO: 26), GVWYFDS (HCDR3; SEQ ID NO:62), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), AASSLQS (LCDR2; SEQ ID NO: 24)and QQYANYPRT (LCDR3; SEQ ID NO: 25) [Clone MH5.23];

(gg) the amino acid sequences GYTFTDFYMN (HCDR1; SEQ ID NO: 180),MGRIYPASGNTYYAQKFQG (HCDR2; SEQ ID NO: 26), GVWYFDT (HCDR3; SEQ ID NO:63), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), AASSLQS (LCDR2; SEQ ID NO: 24)and QQYANYPRT (LCDR3; SEQ ID NO: 25) [Clone MH5.24];

(hh) the amino acid sequences GYTFTNYYMN (HCDR1; SEQ ID NO: 181),MGRIYPASGNTYYAQKFQG (HCDR2; SEQ ID NO: 26), GVWYFDS (HCDR3; SEQ ID NO:62), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), AASSLQS (LCDR2; SEQ ID NO: 24)and QQYANYPRT (LCDR3; SEQ ID NO: 25) [Clone MH5.34];

(ii) the amino acid sequences GYTFTNYYMN (HCDR1; SEQ ID NO: 181),MGRIYPASGNTYYAQKFQG (HCDR2; SEQ ID NO: 26), GVWYFDT (HCDR3; SEQ ID NO:63), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), AASSLQS (LCDR2; SEQ ID NO: 24)and QQYANYPRT (LCDR3; SEQ ID NO: 25) [Clone MH5.35];

(jj) the amino acid sequences GYTFTDYYMN (HCDR1; SEQ ID NO: 13),MGRIYPGSGNTYYAQKFQG (HCDR2; SEQ ID NO: 14), GVYYYDY (HCDR3; SEQ ID NO:15), RASQGIRTNLA (LCDR1; SEQ ID NO: 16), AASSLQS (LCDR2; SEQ ID NO: 24)and QQYANYPRT (LCDR3; SEQ ID NO: 25) [Clone MH5-DI].

In some aspects, disclosed herein is an anti-C-KIT antibody or anantigen-binding portion thereof, wherein the antibody comprises a heavychain variable (VH) region and a light chain variable (VL) region,wherein

-   -   (a) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYYDY (SEQ ID NO: 15); and the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNLA (SEQ ID NO:        16), LCDR2 of AASSLQS (SEQ ID NO: 24) and LCDR3 of QQYNSYPRT        (SEQ ID NO: 12);    -   (b) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYYDY (SEQ ID NO: 15); and the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNLA (SEQ ID NO:        16), LCDR2 of AASSLQS (SEQ ID NO: 24) and LCDR3 of QQYANYPRT        (SEQ ID NO: 25);    -   (c) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYYDY (SEQ ID NO: 15); and the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNLA (SEQ ID NO:        16), LCDR2 of SASSLQS (SEQ ID NO: 17) and LCDR3 of QQYANYPRT        (SEQ ID NO: 25);    -   (d) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYYDY (SEQ ID NO: 15); and the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNLA (SEQ ID NO:        16), LCDR2 of SASSLQS (SEQ ID NO: 17) and LCDR3 of QQYNSYPRT        (SEQ ID NO: 12);    -   (e) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYLDY (SEQ ID NO: 18); and the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNVA (SEQ ID NO:        19), LCDR2 of SASYRQS (SEQ ID NO: 20) and LCDR3 of QQYNSYPRT        (SEQ ID NO: 12);    -   (f) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYFDE (SEQ ID NO: 21); and the VL region        amino acid sequence comprises LCDR1 of RASQGVRTNLA (SEQ ID NO:        22), LCDR2 of AASSRQS (SEQ ID NO: 23) and LCDR3 of QQYNSYPRT        (SEQ ID NO: 12); or    -   (g) the VH region amino acid sequence comprises HCDR1 of        GYTFTDFYMN (SEQ ID NO: 180), HCDR2 of MGRIYPASGNTYYAQKFQG (SEQ        ID NO: 26) and HCDR3 of GVWYYDY (SEQ ID NO: 27); and the VL        region amino acid sequence comprises LCDR1 of RASQGIRTNLA (SEQ        ID NO: 16), LCDR2 of AASSLQS (SEQ ID NO: 24) and LCDR3 of        QQYANYPRT (SEQ ID NO: 25).

In some aspects, disclosed herein is an anti-C-KIT antibody or anantigen-binding portion thereof, wherein the antibody comprises a heavychain variable (VH) region and a light chain variable (VL) region,wherein the VH region comprises any one of the VH region amino acidsequences in Table 12 and the VL region comprises any one of the VLregion amino acid sequences in Table 12.

In some aspects, disclosed herein is an anti-C-KIT antibody or anantigen-binding portion thereof, wherein the antibody comprises a heavychain variable (VH) region and a light chain variable (VL) region,wherein

-   -   (a) the VH region amino acid sequence comprises or consists of        SEQ ID NO:185 and the VL region amino acid sequence comprises or        consists of SEQ ID NO:186;    -   (b) the VH region amino acid sequence comprises or consists of        SEQ ID NO:187 and the VL region amino acid sequence comprises or        consists of SEQ ID NO:188;    -   (c) the VH region amino acid sequence comprises or consists of        SEQ ID NO:189 and the VL region amino acid sequence comprises or        consists of SEQ ID NO:190;    -   (d) the VH region amino acid sequence comprises or consists of        SEQ ID NO:191 and the VL region amino acid sequence comprises or        consists of SEQ ID NO:192;    -   (e) the VH region amino acid sequence comprises or consists of        SEQ ID NO:193 and the VL region amino acid sequence comprises or        consists of SEQ ID NO:194; or    -   (f) the VH region amino acid sequence comprises or consists of        SEQ ID NO:195 and the VL region amino acid sequence comprises or        consists of SEQ ID NO:196.

In some aspects, disclosed herein is an anti-C-KIT antibody or anantigen-binding portion thereof, wherein the antibody comprises a heavychain variable (VH) region and a light chain variable (VL) region,wherein

-   -   (a) the VH region amino acid sequence is at least about 90%, at        least about 91%, at least about 92%, at least about 93%, at        least about 94%, at least about 95%, at least about 96%, at        least about 97%, at least about 98% or at least about 99%        identical to SEQ ID NO:185 and the VL region amino acid sequence        is at least about 90%, at least about 91%, at least about 92%,        at least about 93%, at least about 94%, at least about 95%, at        least about 96%, at least about 97%, at least about 98% or at        least about 99% identical to SEQ ID NO:186;    -   (b) the VH region amino acid sequence is at least about 90%, at        least about 91%, at least about 92%, at least about 93%, at        least about 94%, at least about 95%, at least about 96%, at        least about 97%, at least about 98% or at least about 99%        identical to SEQ ID NO:187 and the VL region amino acid sequence        is at least about 90%, at least about 91%, at least about 92%,        at least about 93%, at least about 94%, at least about 95%, at        least about 96%, at least about 97%, at least about 98% or at        least about 99% identical to SEQ ID NO:188;    -   (c) the VH region amino acid sequence is at least about 90%, at        least about 91%, at least about 92%, at least about 93%, at        least about 94%, at least about 95%, at least about 96%, at        least about 97%, at least about 98% or at least about 99%        identical to SEQ ID NO:189 and the VL region amino acid sequence        is at least about 90%, at least about 91%, at least about 92%,        at least about 93%, at least about 94%, at least about 95%, at        least about 96%, at least about 97%, at least about 98% or at        least about 99% identical to SEQ ID NO:190;    -   (d) the VH region amino acid sequence is at least about 90%, at        least about 91%, at least about 92%, at least about 93%, at        least about 94%, at least about 95%, at least about 96%, at        least about 97%, at least about 98% or at least about 99%        identical to SEQ ID NO:191 and the VL region amino acid sequence        is at least about 90%, at least about 91%, at least about 92%,        at least about 93%, at least about 94%, at least about 95%, at        least about 96%, at least about 97%, at least about 98% or at        least about 99% identical to SEQ ID NO:192;    -   (e) the VH region amino acid sequence is at least about 90%, at        least about 91%, at least about 92%, at least about 93%, at        least about 94%, at least about 95%, at least about 96%, at        least about 97%, at least about 98% or at least about 99%        identical to SEQ ID NO:193 and the VL region amino acid sequence        is at least about 90%, at least about 91%, at least about 92%,        at least about 93%, at least about 94%, at least about 95%, at        least about 96%, at least about 97%, at least about 98% or at        least about 99% identical to SEQ ID NO:194; or    -   (f) the VH region amino acid sequence is at least about 90%, at        least about 91%, at least about 92%, at least about 93%, at        least about 94%, at least about 95%, at least about 96%, at        least about 97%, at least about 98% or at least about 99%        identical to SEQ ID NO:195 and the VL region amino acid sequence        is at least about 90%, at least about 91%, at least about 92%,        at least about 93%, at least about 94%, at least about 95%, at        least about 96%, at least about 97%, at least about 98% or at        least about 99% identical to SEQ ID NO:196. In some aspects, the        CDR amino acid sequences of an anti-C-KIT antibody are 100%        identical to the CDR amino acid sequences in the recited        sequences while the FR amino acid sequences are less than 100%        identical to the FR amino acid sequences in the recited        sequences.

In some aspects, the antibody or antigen-binding portion as definedherein may be isolated.

The antibody molecule or antigen-binding portion as defined herein maycross-compete for binding to C-KIT with an antibody or antigen-bindingportion thereof comprising the sets of CDRs disclosed herein. In someembodiments, the invention provides an isolated anti-C-KIT antibody oran antigen-binding portion thereof, wherein the antibody orantigen-binding portion cross-competes for binding to C-KIT with theantibody or antigen-binding portion comprising the sets of CDRsdisclosed herein and (a) comprises fully germline human framework aminoacid sequences; and/or (b) does not comprise a deamidation site in theLCDR3; and/or (c) comprises a human germline peptide sequence with highMHC class II binding affinity in HCDR1 and/or LCDR2; and/or (d)comprises a reduced number of immunogenic peptides compared to ananti-C-KIT antibody comprising the variable domain sequences of antibodyh37M (Table 2); and/or (e) exhibits reduced immunogenicity compared toan anti-C-KIT antibody comprising the variable domain sequences ofantibody h37M (Table 2); and/or (f) exhibits similar potency in receptorinternalization to an anti-C-KIT antibody comprising the variable domainsequences of antibody h37M (Table 2) and exhibits reduced potency inC-KIT signalling blockade to an anti-C-KIT antibody comprising thevariable domain sequences of antibody h37M (Table 2); and/or (g) isimmune effector null.

In some embodiments, an anti-C-KIT antibody or antigen-binding portionhas low immunogenicity. In certain cases, an antibody or antigen-bindingportion exhibits reduced immunogenicity compared to an anti-C-KITantibody comprising HCDR1 of GYTFTDYYIN (SEQ ID NO: 4), HCDR2 ofIARIYPGSGNTYYNEKFKG (SEQ ID NO: 5), HCDR3 of GVYYFDY (SEQ ID NO: 6),LCDR1 of KASQNVRTNVA (SEQ ID NO: 10), and LCDR2 of SASYRYS (SEQ ID NO:11). In some examples, immunogenicity risk of an antibody orantigen-binding portion may be determined in silico by identifying thelocation of T cell epitopes in the antibody or portion (e.g., in thevariable regions of the antibody or portion).

For example, T cell epitopes in an antibody or antigen-binding portionmay be identified by using iTope™. iTope™ can used to analyse VL and VHregion sequences for peptides with promiscuous high affinity binding tohuman MHC class II. Promiscuous high affinity MHC class II bindingpeptides are thought to correlate with the presence of T cell epitopesthat are high risk indicators for clinical immunogenicity of drugproteins. The iTope™ software predicts favourable interactions betweenamino acid side chains of a peptide and specific binding pockets (inparticular pocket positions; p1, p4, p6, p7 and p9) within theopen-ended binding grooves of 34 human MHC class II alleles. Thesealleles represent the most common HLA-DR alleles found world-wide withno weighting attributed to those found most prevalently in anyparticular ethnic population. Twenty of the alleles contain the ‘open’p1 configuration and 14 contain the ‘closed’ configuration where glycineat position 83 is replaced by a valine. The location of key bindingresidues is achieved by the in silico generation of 9 mer peptides thatoverlap by eight amino acids spanning the test protein sequence. Thisprocess successfully discriminates with high accuracy between peptidesthat either bind or do not bind MHC class II molecules.

T cell epitopes in an antibody or antigen-binding portion may beidentified by analysing VL and VH region sequences using TCED™ (T CellEpitope Database™) to search for matches to T cell epitopes previouslyidentified by in vitro human T cell epitope mapping analyses of otherprotein sequences. The TCED™ is used to search any test sequence againsta large (>10,000 peptides) database of peptides derived from unrelatedprotein and antibody sequences.

In some embodiments, an anti-C-KIT antibody or antigen-binding portionmay exhibit a low immunogenicity because the antibody or portion has alow number of one or more of the following peptides in its sequences:High Affinity Foreign (‘HAF’—high immunogenicity risk), Low AffinityForeign (‘LAF’—lower immunogenicity risk), and/or TCED+(previouslyidentified epitope in TCED™ database).

In some embodiments, an anti-C-KIT antibody or antigen-binding portionmay have high Germline Epitope (GE) content in its sequence. In someexamples, an anti-C-KIT antibody or antigen-binding portion has 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 (or greater than 20) germlineepitopes in its sequence. Germline Epitope may be defined as a humangermline peptide sequence with high MHC Class II binding affinity.Germline Epitope 9 mer peptides are unlikely to have immunogenicpotential due to T cell tolerance, as validated by previous studies witha wide range of germline peptides. Importantly, such germline v-domainepitopes (aided further by similar sequences in the human antibodyconstant regions) also compete for MHC Class II occupancy at themembrane of antigen presenting cells, reducing the risk of foreignpeptide presentation being sufficient to achieve the ‘activationthreshold’ required for T cell stimulation. High GE content is thereforea beneficial quality in clinical development of an antibody therapeuticand can provide low immunogenicity. In some examples, an anti-C-KITantibody or antigen-binding portion comprises a human germline peptidesequence with high MHC class II binding affinity (e.g., germlineepitope) in the HCDR1 and/or LCDR2.

In certain embodiments, an anti-C-KIT antibody or antigen-bindingportion may have a reduced number of HAF, LAF and/or TCED+ epitopesfound in the frameworks of both the heavy and light chain variableregions compared to an anti-C-KIT antibody comprising the variabledomain sequences of antibody h37M (Table 2). For example, a TCED+ andHAF peptide ‘VTITCKASQ’ (SEQ ID NO: 64) found in the LCDR-1 of h37M maybe eliminated in an anti-C-KIT antibody or antigen-binding portion bythe mutation K>R at position 6, converting this sequence to the lightchain GE ‘VTITCRASQ’ (SEQ ID NO: 65; FIG. 11B-H). Similarly, one or bothof the HAF peptides ‘LIYSASSLQ’ (SEQ ID NO: 66) and ‘IYSASSLQS’ (SEQ IDNO: 67) may be converted to GE sequences by mutation of the LCDR2sequence to the fully germline sequence ‘AASSLQS’ (SEQ ID NO: 24).

In one embodiment, an anti-C-KIT antibody or antigen-binding portioncomprises the HCDR1 germlining mutation I>M at position 3 of the TCED+and HAF peptide sequence ‘YYINWVRQA’ (SEQ ID NO: 68; spanning the HCDR-1and FW2).

In some embodiments, an anti-C-KIT antibody or antigen-binding portioncomprises the mutation Y>W in the HCDR3 that eliminates two LAF peptidesequences found in antibody h37M.

The terms “cross-compete”, “cross-competition”, “cross-block”,“cross-blocked” and “cross-blocking” are used interchangeably herein tomean the ability of an antibody or portion thereof to interfere with thebinding directly or indirectly through allosteric modulation of theanti-C-KIT antibodies of the invention to the target C-KIT (e.g., humanC-KIT). The extent to which an antibody or portion thereof is able tointerfere with the binding of another to the target, and thereforewhether it can be said to cross-block or cross-compete according to theinvention, can be determined using competition binding assays. Oneexample of a binding competition assay is Homogeneous Time ResolvedFluorescence (HTRF). One particularly suitable quantitativecross-competition assay uses a FACS- or an AlphaScreen-based approach tomeasure competition between the labelled (e.g. His tagged, biotinylatedor radioactive labelled) antibody or portion thereof and the otherantibody or portion thereof in terms of their binding to the target. Ingeneral, a cross-competing antibody or portion thereof is, for example,one which will bind to the target in the cross-competition assay suchthat, during the assay and in the presence of a second antibody orportion thereof, the recorded displacement of the immunoglobulin singlevariable domain or polypeptide according to the invention is up to 100%(e.g. in a FACS based competition assay) of the maximum theoreticaldisplacement (e.g. displacement by cold (e.g. unlabeled) antibody orfragment thereof that needs to be cross-blocked) by the potentiallycross-blocking antibody or fragment thereof that is present in a givenamount. Preferably, cross-competing antibodies or portions thereof havea recorded displacement that is between 10% and 100%, or between 50% and100%.

The antibody molecule or antigen-binding portion as defined herein maycomprise one or more substitutions, deletions and/or insertions whichremove a post-translational modification (PTM) site, for example aglycosylation site (N-linked or O-linked), a deamination site, aphosphorylation site or an isomerisation/fragmentation site.

More than 350 types of PTM are known. Key forms of PTM includephosphorylation, glycosylation (N- and O-linked), sumoylation,palmitoylation, acetylation, sulfation, myristoylation, prenylation andmethylation (of K and R residues). Statistical methods to identifyputative amino acid sites responsible for specific PTMs are well knownin the art (see Zhou et al., 2016, Nature Protocols 1: 1318-1321).Removal of such a site for example by substitution, deletion and/orinsertion and then optionally testing (experimentally and/ortheoretically) for (a) binding activity and/or (b) loss of the PTM iscontemplated.

For example, the 37M murine LCDR3 (as defined herein, i.e. the aminoacid sequence QQYNSYPRT (SEQ ID NO: 12) has been identified to have aputative deamidation site at residue 4 (N). Removal this site atequivalent positions in an LCDR3 of the invention, for example bysubstitution (such as to A, S, E or T), is envisaged (as for example inclone MH5 and mutant derivatives of MH5, as in Tables 4 and 6).

The antibody molecule or antigen-binding portion thereof may be human,humanized or chimeric.

The antibody molecule or antigen-binding portion thereof may compriseone or more human variable domain framework scaffolds into which theCDRs have been inserted. For example, the VH region, the VL region, orboth the VH and the VL region may comprise one or more human frameworkregion (FR) amino acid sequences.

The antibody molecule or antigen-binding portion thereof may comprise anIGHV1-46 human germline scaffold into which the corresponding HCDRsequences have been inserted. The antibody molecule or antigen-bindingportion thereof may comprise a VH region that comprises an IGHV1-46human germline scaffold amino acid sequence into which a set ofcorresponding HCDR1, HCDR2 and HCDR3 amino acid sequences have beeninserted.

The antibody molecule or antigen-binding portion thereof may comprise anIGKV1-16 human germline scaffold into which the corresponding LCDRsequences have been inserted. The antibody molecule or antigen-bindingportion thereof may comprise a VL region that comprises an IGKV1-16human germline scaffold amino acid sequence into which a set ofcorresponding LCDR1, LCDR2 and LCDR3 amino acid sequences have beeninserted.

The antibody molecule or antigen-binding portion thereof may comprise anIGHV1-46 human germline scaffold into which the corresponding HCDRsequences have been inserted and an IGKV1-16 human germline scaffoldinto which the corresponding LCDR sequences have been inserted. Theantibody molecule or antigen-binding portion thereof may comprise a VHregion that comprises an IGHV1-46 human germline scaffold amino acidsequence into which a set of corresponding HCDR1, HCDR2 and HCDR3 aminoacid sequences have been inserted and a VL region that comprises anIGKV1-16 human germline scaffold amino acid sequence into which a set ofcorresponding LCDR1, LCDR2 and LCDR3 amino acid sequences have beeninserted. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acidsequences may be the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 aminoacid sequences of any one of the clones in Table 4 or Table 6 (with allsix CDR sequences being from the same clone).

In some aspects, the antibody molecule or antigen-binding portionthereof may comprise an immunoglobulin constant region. In some aspects,the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1 orIgA2. In additional aspects, the immunoglobulin constant region is IgG1,IgG2, IgG3, IgG4, IgA1 or IgA2. The antibody molecule or antigen-bindingportion thereof may comprise an immunologically inert constant region.In some aspects, an anti-C-KIT antibody or antigen-binding portionthereof may comprise an immunoglobulin constant region comprising awild-type human IgG1 constant region, a human IgG1 constant regioncomprising the amino acid substitutions L234A, L235A and G237A or ahuman IgG1 constant region comprising the amino acid substitutionsL234A, L235A, G237A and P331S. In some aspects, an anti-C-KIT antibodymay comprise an immunoglobulin constant region comprising any one of theamino acid sequences in Table 13. The Fc region sequences in Table 13begin at the CH1 domain. In some aspects, an anti-C-KIT antibody maycomprise an immunoglobulin constant region comprising an amino acidsequence of an Fc region of human IgG1, human IgG1-3M or human IgG1-4M.For example, the human IgG1-3M Fc region comprises the followingsubstitutions: L234A, L235A and G237A, while the human IgG1-4M Fc regioncomprises the following substitutions: L234A, L235A, G237A and P331S. Insome aspects, a position of an amino acid residue in a constant regionof an immunoglobulin molecule is numbered according to EU nomenclature(Ward et al., 1995 Therap. Immunol. 2:77-94). In some aspects, animmunoglobulin constant region may comprise an RDELT (SEQ ID NO:203)motif or an REEM (SEQ ID NO:204) motif (underlined in Table 13). TheREEM (SEQ ID NO:204) allotype is found in a smaller human populationthan the RDELT (SEQ ID NO:203) allotype. In some aspects, an anti-C-KITantibody may comprise an immunoglobulin constant region comprising anyone of SEQ ID NOs:197-202. In some aspects, an anti-C-KIT antibody maycomprise the six CDR amino acid sequences of any one of the clones inTable 4 or 6 and any one of the Fc region amino acid sequences in Table13. In some aspects, an anti-C-KIT antibody may comprise animmunoglobulin heavy chain constant region comprising any one of the Fcregion amino acid sequences in Table 13 and an immunoglobulin lightchain constant region that is a kappa light chain constant region or alambda light chain constant region.

In some aspects, disclosed herein is an anti-C-KIT antibody or anantigen-binding portion thereof, wherein the antibody comprises a heavychain variable (VH) region, a light chain variable (VL) region and aheavy chain constant region, wherein

-   -   (a) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYYDY (SEQ ID NO: 15); the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNLA (SEQ ID NO:        16), LCDR2 of AASSLQS (SEQ ID NO: 24) and LCDR3 of QQYNSYPRT        (SEQ ID NO: 12); and the heavy chain constant region comprises        SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ        ID NO:201 or SEQ ID NO:202;    -   (b) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYYDY (SEQ ID NO: 15); the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNLA (SEQ ID NO:        16), LCDR2 of AASSLQS (SEQ ID NO: 24) and LCDR3 of QQYANYPRT        (SEQ ID NO: 25); and the heavy chain constant region comprises        SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ        ID NO:201 or SEQ ID NO:202;    -   (c) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYYDY (SEQ ID NO: 15); the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNLA (SEQ ID NO:        16), LCDR2 of SASSLQS (SEQ ID NO: 17) and LCDR3 of QQYANYPRT        (SEQ ID NO: 25); and the heavy chain constant region comprises        SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ        ID NO:201 or SEQ ID NO:202;    -   (d) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYYDY (SEQ ID NO: 15); the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNLA (SEQ ID NO:        16), LCDR2 of SASSLQS (SEQ ID NO: 17) and LCDR3 of QQYNSYPRT        (SEQ ID NO: 12); and the heavy chain constant region comprises        SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ        ID NO:201 or SEQ ID NO:202;    -   (e) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYLDY (SEQ ID NO: 18); the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNVA (SEQ ID NO:        19), LCDR2 of SASYRQS (SEQ ID NO: 20) and LCDR3 of QQYNSYPRT        (SEQ ID NO: 12); and the heavy chain constant region comprises        SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ        ID NO:201 or SEQ ID NO:202;    -   (f) the VH region amino acid sequence comprises HCDR1 of        GYTFTDYYMN (SEQ ID NO: 13), HCDR2 of MGRIYPGSGNTYYAQKFQG (SEQ ID        NO: 14) and HCDR3 of GVYYFDE (SEQ ID NO: 21); the VL region        amino acid sequence comprises LCDR1 of RASQGVRTNLA (SEQ ID NO:        22), LCDR2 of AASSRQS (SEQ ID NO: 23) and LCDR3 of QQYNSYPRT        (SEQ ID NO: 12); and the heavy chain constant region comprises        SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ        ID NO:201 or SEQ ID NO:202; or    -   (g) the VH region amino acid sequence comprises HCDR1 of        GYTFTDFYMN (SEQ ID NO: 180), HCDR2 of MGRIYPASGNTYYAQKFQG (SEQ        ID NO: 26) and HCDR3 of GVWYYDY (SEQ ID NO: 27); the VL region        amino acid sequence comprises LCDR1 of RASQGIRTNLA (SEQ ID NO:        16), LCDR2 of AASSLQS (SEQ ID NO: 24) and LCDR3 of QQYANYPRT        (SEQ ID NO: 25); and the heavy chain constant region comprises        SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ        ID NO:201 or SEQ ID NO:202.

In some aspects, disclosed herein is an anti-C-KIT antibody or anantigen-binding portion thereof, wherein the antibody comprises a heavychain variable (VH) region, a light chain variable (VL) region and aheavy chain constant region, wherein

-   -   (a) the VH region amino acid sequence comprises or consists of        SEQ ID NO:185, the VL region amino acid sequence comprises or        consists of SEQ ID NO:186 and the heavy chain constant region        comprises a wild-type human IgG1 constant region, a human IgG1        constant region comprising the amino acid substitutions L234A,        L235A and G237A or a human IgG1 constant region comprising the        amino acid substitutions L234A, L235A, G237A and P331S;    -   (b) the VH region amino acid sequence comprises or consists of        SEQ ID NO:187, the VL region amino acid sequence comprises or        consists of SEQ ID NO:188 and the heavy chain constant region        comprises a wild-type human IgG1 constant region, a human IgG1        constant region comprising the amino acid substitutions L234A,        L235A and G237A or a human IgG1 constant region comprising the        amino acid substitutions L234A, L235A, G237A and P331S;    -   (c) the VH region amino acid sequence comprises or consists of        SEQ ID NO:189, the VL region amino acid sequence comprises or        consists of SEQ ID NO:190 and the heavy chain constant region        comprises a wild-type human IgG1 constant region, a human IgG1        constant region comprising the amino acid substitutions L234A,        L235A and G237A or a human IgG1 constant region comprising the        amino acid substitutions L234A, L235A, G237A and P331S;    -   (d) the VH region amino acid sequence comprises or consists of        SEQ ID NO:191, the VL region amino acid sequence comprises or        consists of SEQ ID NO:192 and the heavy chain constant region        comprises a wild-type human IgG1 constant region, a human IgG1        constant region comprising the amino acid substitutions L234A,        L235A and G237A or a human IgG1 constant region comprising the        amino acid substitutions L234A, L235A, G237A and P331S;    -   (e) the VH region amino acid sequence comprises or consists of        SEQ ID NO:193, the VL region amino acid sequence comprises or        consists of SEQ ID NO:194 and the heavy chain constant region        comprises a wild-type human IgG1 constant region, a human IgG1        constant region comprising the amino acid substitutions L234A,        L235A and G237A or a human IgG1 constant region comprising the        amino acid substitutions L234A, L235A, G237A and P331S; or    -   (f) the VH region amino acid sequence comprises or consists of        SEQ ID NO:195, the VL region amino acid sequence comprises or        consists of SEQ ID NO:196 and the heavy chain constant region        comprises a wild-type human IgG1 constant region, a human IgG1        constant region comprising the amino acid substitutions L234A,        L235A and G237A or a human IgG1 constant region comprising the        amino acid substitutions L234A, L235A, G237A and P331S.

In some aspects, disclosed herein is an anti-C-KIT antibody or anantigen-binding portion thereof, wherein the antibody comprises a heavychain variable (VH) region, a light chain variable (VL) region and aheavy chain constant region, wherein

-   -   (a) the VH region amino acid sequence comprises or consists of        SEQ ID NO:185, the VL region amino acid sequence comprises or        consists of SEQ ID NO:186 and the heavy chain constant region        comprises SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID        NO:200, SEQ ID NO:201 or SEQ ID NO:202;    -   (b) the VH region amino acid sequence comprises or consists of        SEQ ID NO:187, the VL region amino acid sequence comprises or        consists of SEQ ID NO:188 and the heavy chain constant region        comprises SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID        NO:200, SEQ ID NO:201 or SEQ ID NO:202;    -   (c) the VH region amino acid sequence comprises or consists of        SEQ ID NO:189, the VL region amino acid sequence comprises or        consists of SEQ ID NO:190 and the heavy chain constant region        comprises SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID        NO:200, SEQ ID NO:201 or SEQ ID NO:202;    -   (d) the VH region amino acid sequence comprises or consists of        SEQ ID NO:191, the VL region amino acid sequence comprises or        consists of SEQ ID NO:192 and the heavy chain constant region        comprises SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID        NO:200, SEQ ID NO:201 or SEQ ID NO:202;    -   (e) the VH region amino acid sequence comprises or consists of        SEQ ID NO:193, the VL region amino acid sequence comprises or        consists of SEQ ID NO:194 and the heavy chain constant region        comprises SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID        NO:200, SEQ ID NO:201 or SEQ ID NO:202; or    -   (f) the VH region amino acid sequence comprises or consists of        SEQ ID NO:195, the VL region amino acid sequence comprises or        consists of SEQ ID NO:196 and the heavy chain constant region        comprises SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID        NO:200, SEQ ID NO:201 or SEQ ID NO:202.

In some aspects, an anti-C-KIT antibody may be immune effector null. Insome aspects, an anti-C-KIT antibody or an antigen-binding portionthereof does not induce immune effector function and, optionally,suppresses immune effector function. In some aspects, an anti-C-KITantibody may lack measurable binding to human FcγRI, FcγRIIa, FcγRIIIaand FcγRIIIb receptors but maintain binding to human FcγRIIb receptorand optionally maintain binding to human FcRn receptor. FcγRI, FcγRIIa,FcγRIIIa and FcγRIIIb are examples of activating receptors. FcγRIIb isan example of an inhibitory receptor. FcRn is an example of a recyclingreceptor. In some aspects, binding affinity of an anti-C-KIT antibody oran antigen-binding portion thereof for human Fc receptors may bemeasured by BIACORE® analysis. In some aspects, Homogeneous TimeResolved Fluorescence (HTRF) can be used to study binding of ananti-C-KIT antibody to human Fc receptors. In one example of HTRF, humanIgG1 (wild type) is labelled, as is the full suite of Fc gamma receptorsand then antibodies with engineered Fc fragments are used in titrationcompetition. In some aspects, KIT-positive cells may be mixed with humanwhite blood cells and anti-C-KIT antibodies, and cell killing by CDC,ADCC and/or ADCP may be measured. In some aspects, an anti-C-KITantibody may comprise an amino acid sequence of an Fc region of humanIgG1-3M or human IgG1-4M (see Table 13) is effector null. In someaspects, an anti-C-KIT antibody may comprise an amino acid sequence ofan Fc region of human IgG1-3M or human IgG1-4M (see Table 13) is noteffector null.

The antibody molecule or antigen-binding portion thereof may be a Fabfragment, a F(ab)₂ fragment, an Fv fragment, a tetrameric antibody, atetravalent antibody, a multispecific antibody (for example, abispecific antibody), a domain-specific antibody, a single domainantibody, a monoclonal antibody or a fusion protein. In one embodiment,an antibody may be a bispecific antibody that binds specifically to afirst antigen and a second antigen, wherein the first antigen is C-KITand the second antigen is not C-KIT. Antibody molecules and methods fortheir construction and use are described, in for example Holliger &Hudson (2005, Nature Biotechnol. 23(9): 1126-1136).

In another aspect of the invention, there is provided an immunoconjugatecomprising the antibody molecule or antigen-binding portion thereof ofthe invention as defined herein linked to a therapeutic agent.

Examples of suitable therapeutic agents include cytotoxins,radioisotopes, chemotherapeutic agents, immunomodulatory agents,anti-angiogenic agents, antiproliferative agents, pro-apoptotic agents,and cytostatic and cytolytic enzymes (for example RNAses). Furthertherapeutic agents include a therapeutic nucleic acid, such as a geneencoding an immunomodulatory agent, an anti-angiogenic agent, ananti-proliferative agent, or a pro-apoptotic agent. These drugdescriptors are not mutually exclusive, and thus a therapeutic agent maybe described using one or more of the above terms.

Examples of suitable therapeutic agents for use in immunoconjugatesinclude the taxanes, maytansines, CC-1065 and the duocarmycins, thecalicheamicins and other enediynes, and the auristatins. Other examplesinclude the anti-folates, vinca alkaloids, and the anthracyclines. Planttoxins, other bioactive proteins, enzymes (i.e., ADEPT), radioisotopes,photosensitizers may also be used in immunoconjugates. In addition,conjugates can be made using secondary carriers as the cytotoxic agent,such as liposomes or polymers, Suitable cytotoxins include an agent thatinhibits or prevents the function of cells and/or results in destructionof cells. Representative cytotoxins include antibiotics, inhibitors oftubulin polymerization, alkylating agents that bind to and disrupt DNA,and agents that disrupt protein synthesis or the function of essentialcellular proteins such as protein kinases, phosphatases, topoisomerases,enzymes, and cyclins.

Representative cytotoxins include, but are not limited to, doxorubicin,daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone,epirubicin, carubicin, nogalamycin, menogaril, pitarubicin, valrubicin,cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine,azacitidine, doxifluhdine, pentostatin, broxuhdine, capecitabine,cladhbine, decitabine, floxuhdine, fludarabine, gougerotin, puromycin,tegafur, tiazofuhn, adhamycin, cisplatin, carboplatin, cyclophosphamide,dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin,mechlorethamine, prednisone, procarbazine, methotrexate, flurouracils,etoposide, taxol, taxol analogs, platins such as cis-platin andcarbo-platin, mitomycin, thiotepa, taxanes, vincristine, daunorubicin,epirubicin, actinomycin, authramycin, azaserines, bleomycins, tamoxifen,idarubicin, dolastatins/auristatins, hemiasterlins, esperamicins andmaytansinoids.

Suitable immunomodulatory agents include anti-hormones that blockhormone action on tumors and immunosuppressive agents that suppresscytokine production, down-regulate self-antigen expression, or mask MHCantigens.

Also provided is a nucleic acid molecule encoding the antibody moleculeor antigen-binding portion thereof of the invention as defined herein.An isolated nucleic acid molecule may encode (a) the VH region aminoacid sequence; (b) the VL region amino acid sequence; or (c) both the VHand the VL region amino acid sequences of an anti-C-KIT antibody or anantigen-binding portion thereof described herein.

Further provided is a vector comprising the nucleic acid molecule of theinvention as defined herein. The vector may be an expression vector. Thevector may further comprise one or more regulatory sequences (e.g., apromoter and/or an enhancer).

Also provided is a host cell comprising the nucleic acid molecule or thevector of the invention as defined herein. The host cell may be arecombinant host cell.

In a further aspect there is provided a method of producing ananti-C-KIT antibody and/or an antigen-binding portion thereof,comprising culturing the host cell of the invention under conditionsthat result in expression and/or production of the antibody and/or theantigen-binding portion thereof, and isolating the antibody and/or theantigen-binding portion thereof from the host cell or culture.

In another aspect of the invention there is provided a pharmaceuticalcomposition comprising the antibody molecule or antigen-binding portionthereof of the invention as defined herein, or the nucleic acid moleculeof the invention as defined herein, or the vector of the invention asdefined herein.

Further provided is a method for enhancing an immune response in asubject, comprising administering an effective amount of the antibodymolecule or antigen-binding portion thereof of the invention as definedherein, or the immunoconjugate of the invention as defined herein, orthe nucleic acid molecule of the invention as defined herein, or thevector of the invention as defined herein, or the pharmaceuticalcomposition of the invention as defined herein.

In a further aspect there is provided a method for treating orpreventing cancer in a subject, comprising administering an effectiveamount of the antibody molecule or antigen-binding portion thereof ofthe invention as defined herein, or the immunoconjugate of the inventionas defined herein, or the nucleic acid molecule of the invention asdefined herein, or the vector of the invention as defined herein, or thepharmaceutical composition of the invention as defined herein.

The cancer may for example be selected from the group consisting of:Gastrointestinal Stromal cancer (GIST), pancreatic cancer, melanoma,breast cancer, lung cancer, bronchial cancer, colorectal cancer,prostate cancer, stomach cancer, ovarian cancer, urinary bladder cancer,brain or central nervous system cancer, peripheral nervous systemcancer, esophageal cancer, cervical cancer, uterine or endometrialcancer, cancer of the oral cavity or pharynx, liver cancer, kidneycancer, testicular cancer, biliary tract cancer, small bowel or appendixcancer, salivary gland cancer, thyroid gland cancer, adrenal glandcancer, osteosarcoma, chondrosarcoma, and cancer of hematologicaltissues.

The invention also provides an antibody molecule or antigen-bindingportion thereof of the invention as defined herein, or theimmunoconjugate of the invention as defined herein, or the nucleic acidmolecule of the invention as defined herein, or the vector of theinvention as defined herein, or the pharmaceutical composition of theinvention as defined herein, for use in the treatment of cancer.

In another aspect the invention provides the antibody molecule, orantigen-binding portion thereof, or the immunoconjugate, or the nucleicacid molecule, or the vector for use, or the method of treatment of theinvention as defined herein, for separate, sequential or simultaneoususe in a combination combined with a second therapeutic agent, forexample an anti-cancer agent.

In a further aspect there is provided the use of an antibody molecule orantigen-binding portion thereof of the invention as defined herein, oran immunoconjugate of the invention as defined herein, or a nucleic acidmolecule of the invention as defined herein, or a vector of theinvention as defined herein, or a pharmaceutical composition of theinvention as defined herein, in the manufacture of a medicament for thetreatment of cancer.

The invention also provides a method for treating or preventing anautoimmune disease or an inflammatory disease in a subject, comprisingadministering an effective amount of the antibody molecule orantigen-binding portion thereof as defined herein, or theimmunoconjugate as defined here, or the nucleic acid molecule as definedherein, or the vector as defined herein, or the pharmaceuticalcomposition as defined herein.

The autoimmune disease or inflammatory disease may be selected from thegroup consisting of: arthritis, asthma, multiple sclerosis, psoriasis,Crohn's disease, inflammatory bowel disease, lupus, Grave's disease andHashimoto's thyroiditis, and ankylosing spondylitis.

Also provided is an antibody molecule or antigen-binding portion thereofas defined herein, or the immunoconjugate as defined herein, or thenucleic acid molecule as defined herein, or the vector as definedherein, or the pharmaceutical composition as defined herein, for use inthe treatment of an autoimmune disease or an inflammatory disease.

Further provided is the use of an antibody molecule or antigen-bindingportion thereof as defined herein, or an immunoconjugate as definedherein, or a nucleic acid molecule as defined herein, or a vector asdefined herein, or a pharmaceutical composition as defined herein, inthe manufacture of a medicament for the treatment of an autoimmunedisease or an inflammatory disease.

The invention also provides a method for treating or preventing acardiovascular disease or a fibrotic disease in a subject, comprisingadministering an effective amount of the antibody molecule orantigen-binding portion thereof as defined herein, or theimmunoconjugate as defined here, or the nucleic acid molecule as definedherein, or the vector as defined herein, or the pharmaceuticalcomposition as defined herein.

Also provided is an antibody molecule or antigen-binding portion thereofas defined herein, or the immunoconjugate as defined herein, or thenucleic acid molecule as defined herein, or the vector as definedherein, or the pharmaceutical composition as defined herein, for use inthe treatment of a cardiovascular disease or a fibrotic disease.

Further provided is the use of an antibody molecule or antigen-bindingportion thereof as defined herein, or an immunoconjugate as definedherein, or a nucleic acid molecule as defined herein, or a vector asdefined herein, or a pharmaceutical composition as defined herein, inthe manufacture of a medicament for the treatment of a cardiovasculardisease or a fibrotic disease.

The cardiovascular disease in any aspect of the invention may forexample be coronary heart disease or atherosclerosis.

The fibrotic disease in any aspect of the invention may be selected fromthe group consisting of myocardial infarction, angina, osteoarthritis,pulmonary fibrosis, asthma, cystic fibrosis and bronchitis.

In one embodiment, the invention provides an anti-C-KIT antibody or anantigen-binding portion thereof comprising the amino acid sequencesdisclosed herein for use in therapy.

The pharmaceutical composition of the invention may comprise apharmaceutically acceptable excipient, carrier or diluent. Apharmaceutically acceptable excipient may be a compound or a combinationof compounds entering into a pharmaceutical composition which does notprovoke secondary reactions and which allows, for example, facilitationof the administration of the anti-C-KIT antibody molecule, an increasein its lifespan and/or in its efficacy in the body or an increase in itssolubility in solution. These pharmaceutically acceptable vehicles arewell known and will be adapted by the person skilled in the art as afunction of the mode of administration of the anti-C-KIT antibodymolecule.

In some embodiments, the anti-C-KIT antibody molecule may be provided ina lyophilised form for reconstitution prior to administration. Forexample, lyophilised antibody molecules may be re-constituted in sterilewater and mixed with saline prior to administration to an individual.

The anti-C-KIT antibody molecules will usually be administered in theform of a pharmaceutical composition, which may comprise at least onecomponent in addition to the antibody molecule. Thus pharmaceuticalcompositions may comprise, in addition to the anti-C-KIT antibodymolecule, a pharmaceutically acceptable excipient, carrier, buffer,stabilizer or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with theefficacy of the anti-C-KIT antibody molecule. The precise nature of thecarrier or other material will depend on the route of administration,which may be by bolus, infusion, injection or any other suitable route,as discussed below.

For parenteral, for example sub-cutaneous or intra-venousadministration, e.g. by injection, the pharmaceutical compositioncomprising the anti-C-KIT antibody molecule may be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles, such as Sodium Chloride Injection, Ringe's Injection,Lactated Ringer's Injection. Preservatives, stabilizers, buffers,antioxidants and/or other additives may be employed as requiredincluding buffers such as phosphate, citrate and other organic acids;antioxidants, such as ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3′-pentanol; and m-cresol); low molecularweight polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone;amino acids, such as glycine, glutamine, asparagines, histidine,arginine, or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose or dextrins; chelating agents,such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol;salt-forming counter-ions, such as sodium; metal complexes (e.g.Zn-protein complexes); and/or non-ionic surfactants, such as TWEEN™,PLURONICS™ or polyethylene glycol (PEG).

A pharmaceutical composition comprising an anti-C-KIT antibody moleculemay be administered alone or in combination with other treatments,either simultaneously or sequentially dependent upon the condition to betreated.

An anti-C-KIT antibody molecule as described herein may be used in amethod of treatment of the human or animal body, including prophylacticor preventative treatment (e.g. treatment before the onset of acondition in an individual to reduce the risk of the condition occurringin the individual; delay its onset; or reduce its severity after onset).The method of treatment may comprise administering the anti-C-KITantibody molecule to an individual in need thereof.

Administration is normally in a “therapeutically effective amount”, thisbeing sufficient to show benefit to a patient. Such benefit may be atleast amelioration of at least one symptom. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated, the particular mammalbeing treated, the clinical condition of the individual patient, thecause of the disorder, the site of delivery of the composition, themethod of administration, the scheduling of administration and otherfactors known to medical practitioners. Prescription of treatment, e.g.decisions on dosage etc., is within the responsibility of generalpractitioners and other medical doctors and may depend on the severityof the symptoms and/or progression of a disease being treated.Appropriate doses of antibody molecules are well known in the art(Ledermann J. A. et al., 1991, Int. J. Cancer 47: 659-664; Bagshawe K.D. et al., 1991, Antibody, Immunoconjugates and Radiopharmaceuticals 4:915-922). Specific dosages may be indicated herein or in the Physician'sDesk Reference (2003) as appropriate for the type of medicament beingadministered may be used. A therapeutically effective amount or suitabledose of an antibody molecule may be determined by comparing its in vitroactivity and in vivo activity in an animal model. Methods forextrapolation of effective dosages in mice and other test animals tohumans are known. The precise dose will depend upon a number of factors,including whether the antibody is for prevention or for treatment, thesize and location of the area to be treated, the precise nature of theantibody (e.g. whole antibody, fragment) and the nature of anydetectable label or other molecule attached to the antibody.

A typical antibody dose will be in the range 100 μg to 1 g for systemicapplications, and 1 μg to 1 mg for topical applications. An initialhigher loading dose, followed by one or more lower doses, may beadministered. Typically, the antibody will be a whole antibody, e.g. theIgG1 or IgG4 isotype. This is a dose for a single treatment of an adultpatient, which may be proportionally adjusted for children and infants,and also adjusted for other antibody formats in proportion to molecularweight. Treatments may be repeated at daily, twice-weekly, weekly ormonthly intervals, at the discretion of the physician. The treatmentschedule for an individual may be dependent on the pharmocokinetic andpharmacodynamic properties of the antibody composition, the route ofadministration and the nature of the condition being treated.

Treatment may be periodic, and the period between administrations may beabout two weeks or more, e.g. about three weeks or more, about fourweeks or more, about once a month or more, about five weeks or more, orabout six weeks or more. For example, treatment may be every two to fourweeks or every four to eight weeks. Treatment may be given before,and/or after surgery, and/or may be administered or applied directly atthe anatomical site of surgical treatment or invasive procedure.Suitable formulations and routes of administration are described above.

In some embodiments, anti-C-KIT antibody molecules as described hereinmay be administered as sub-cutaneous injections. Sub-cutaneousinjections may be administered using an auto-injector, for example forlong or short-term prophylaxis/treatment.

In some preferred embodiments, the therapeutic effect of the anti-C-KITantibody molecule may persist for several multiples of the antibodyhalf-life in serum, depending on the dose. For example, the therapeuticeffect of a single dose of the anti-C-KIT antibody molecule may persistin an individual for 1 month or more, 2 months or more, 3 months ormore, 4 months or more, 5 months or more, or 6 months or more.

The invention also provides a method of producing an antibody moleculewhich specifically binds to human C-KIT and optionally also tocynomolgus monkey C-KIT or an antigen-binding portion thereof,comprising the steps of:

(1) grafting anti-C-KIT CDRs from a non-human source into a humanv-domain framework to produce a humanized anti-C-KIT antibody moleculeor antigen-binding portion thereof;

(2) generating a phage library of clones of the humanized anti-C-KITantibody molecule or antigen-binding portion thereof comprising one ormore mutations in the CDRs;

(3) selecting the phage library for binding to human C-KIT andoptionally also to cynomolgus monkey C-KIT;

(4) screening clones from the selection step (3) having bindingspecificity to human C-KIT and optionally also to cynomolgus monkeyC-KIT; and

(5) producing an antibody molecule which specifically binds to humanC-KIT and optionally also to cynomolgus monkey C-KIT, or anantigen-binding portion thereof from clones selected from step (4).

The method may comprise a further step of producing additional clonesbased on the clones selected in step (4), for example based on furtherexploratory mutagenesis at specific positions in the CDRs of the clonesselected in step (4), to enhance humanization and/or minimise human Tcell epitope content and/or improve manufacturing properties in theantibody molecule or antigen-binding portion thereof produced in step(5).

Refinements applicable to the above method are as described in Example 1below.

As used herein, the term “C-KIT” refers to CD117 (Cluster ofDifferentiation 117) and variants thereof that retain at least part ofthe biological activity of C-KIT. This protein is also known as KIT,PBT, SCFR and KIT proto-oncogene receptor tyrosine kinase. As usedherein, C-KIT includes all mammalian species of native sequence C-KIT,including human, rat, mouse and chicken. The term “C-KIT” is used toinclude variants, isoforms and species homologs of human C-KIT. Examplesof human C-KIT sequences are provided in Table 14. Antibodies of theinvention may cross-react with C-KIT from species other than human, inparticular C-KIT from cynomolgus monkey (Macaca fascicularis). Incertain embodiments, the antibodies may be completely specific for humanC-KIT and do not exhibit non-human cross-reactivity.

As used herein, an “antagonist” as used in the context of the antibodyof the invention or an “anti-C-KIT antagonist antibody” (interchangeablytermed “anti-C-KIT antibody”) refers to an antibody which is able tobind to C-KIT and inhibit C-KIT biological activity and/or downstreampathway(s) mediated by C-KIT signalling. An anti-C-KIT antagonistantibody encompasses antibodies that can block, antagonize, suppress orreduce (including significantly) C-KIT biological activity, includingdownstream pathways mediated by C-KIT signalling, such as receptorbinding and/or elicitation of a cellular response to C-KIT. For thepurposes of the present invention, it will be explicitly understood thatthe term “anti-C-KIT antagonist antibody” encompass all the terms,titles, and functional states and characteristics whereby C-KIT itself,and C-KIT biological activity (including but not limited to its abilityto enhance the activation of phagocytosis by cells of the myeloidlineage), or the consequences of the activity or biological activity,are substantially nullified, decreased, or neutralized in any meaningfuldegree.

The antibody “specifically binds” “specifically interacts”,“preferentially binds”, “binds” or “interacts” with C-KIT if it bindswith greater affinity, avidity, more readily and/or with greaterduration than it binds to other receptors.

An “antibody molecule” is an immunoglobulin molecule capable of specificbinding to a target, such as a carbohydrate, polynucleotide, lipid,polypeptide, etc., through at least one antigen recognition site,located in the variable region of the immunoglobulin molecule. As usedherein, the term “antibody molecule” encompasses not only intactpolyclonal or monoclonal antibodies, but also any antigen-bindingfragment (for example, an “antigen-binding portion”) or single chainthereof, fusion proteins comprising an antibody, and any other modifiedconfiguration of the immunoglobulin molecule that comprises an antigenrecognition site including, for example without limitation, scFv, singledomain antibodies (for example, shark and camelid antibodies),maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies,v-NAR and bis-scFv.

An “antibody molecule” encompasses an antibody of any class, such asIgG, IgA, or IgM (or sub-class thereof), and the antibody need not be ofany particular class. Depending on the antibody amino acid sequence ofthe constant region of its heavy chains, immunoglobulins can be assignedto different classes. There are five major classes of immunoglobulins:IgA, IgD, IgE, IgG, and IgM, and several of these may be further dividedinto subclasses (isotypes), for example IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2. The heavy-chain constant regions that correspond to the differentclasses of immunoglobulins are called alpha, delta, epsilon, gamma, andmu, respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

The term “antigen-binding portion” of an antibody molecule, as usedherein, refers to one or more fragments of an intact antibody thatretain the ability to specifically bind to C-KIT. Antigen-bindingfunctions of an antibody molecule can be performed by fragments of anintact antibody. Examples of binding fragments encompassed within theterm “antigen binding-portion” of an antibody molecule include Fab;Fab′; F(ab′)₂; an Fd fragment consisting of the VH and CH1 domains; anFv fragment consisting of the VL and VH domains of a single arm of anantibody; a single domain antibody (dAb) fragment, and an isolatedcomplementarity determining region (CDR).

The term “Fc region” is used to define a C-terminal region of animmunoglobulin heavy chain. The “Fc region” may be a native sequence Fcregion or a variant Fc region. Although the boundaries of the Fc regionof an immunoglobulin heavy chain might vary, the human IgG heavy chainFc region is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. Thenumbering of the residues in the Fc region is that of the EU index as inKabat. The Fc region of an immunoglobulin generally comprises twoconstant domains, CH2 and CH3. As is known in the art, an Fc region canbe present in dimer or monomeric form.

A “variable region” or “v-domain” of an antibody refers to the variableregion of the antibody light chain or the variable region of theantibody heavy chain, either alone or in combination. As known in theart, the variable regions of the heavy and light chain each consist offour framework regions (FRs) connected by three complementaritydetermining regions (CDRs) also known as hypervariable regions, andcontribute to the formation of the antigen binding site of antibodies.When choosing FRs to flank CDRs, for example when humanizing oroptimizing an antibody, FRs from antibodies which contain CDR sequencesin the same canonical class are preferred.

The CDR definitions used in the present application combine the domainsused in the many disparate, often conflicting schemes that have beencreated in the field, which are based on the combination ofimmunoglobulin repertoire analyses and structural analyses of antibodiesin isolation and in their co-crystals with antigens (see review bySwindells et al., 2016, abYsis: Integrated Antibody Sequence andStructure-Management, Analysis, and Prediction. J Mol Biol. [PMID:27561707; Epub 22 Aug. 2016]). The CDR definition used herein (a“Unified” definition) incorporates the lessons of all such priorinsights and includes all appropriate loop positions required to samplethe full residue landscape that potentially mediates target-bindingcomplementarity.

Table 1 shows the amino acid sequences of the 37M murine anti-C-KITantibody CDRs as defined herein (a “Unified” scheme), in comparison towell-known alternative systems for defining the same CDRs.

As used herein the term “conservative substitution” refers toreplacement of an amino acid with another amino acid which does notsignificantly deleteriously change the functional activity. A preferredexample of a “conservative substitution” is the replacement of one aminoacid with another amino acid which has a value ≥0 in the followingBLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89:10915-10919):

A R N D C Q E G H I L K M F P S T W Y V A 4 −1 −2 −2 0 −1 −1 0 −2 −1 −1−1 −1 −2 −1 1 0 −3 −2 0 R −1 5 0 −2 −3 1 0 −2 0 −3 −2 2 −1 −3 −2 −1 −1−3 −2 −3 N −2 0 6 1 −3 0 0 0 1 −3 −3 0 −2 −3 −2 1 0 −4 −2 −3 D −2 −2 1 6−3 0 2 −1 −1 −3 −4 −1 −3 −3 −1 0 −1 −4 −3 −3 C 0 −3 −3 −3 9 −3 −4 −3 −3−1 −1 −3 −1 −2 −3 −1 −1 −2 −2 −1 Q −1 1 0 0 −3 5 2 −2 0 −3 −2 1 0 −3 −10 −1 −2 −1 −2 E −1 0 0 2 −4 2 5 −2 0 −3 −3 1 −2 −3 −1 0 −1 −3 −2 −2 G 0−2 0 −1 −3 −2 −2 6 −2 −4 −4 −2 −3 −3 −2 0 −2 −2 −3 −3 H −2 0 1 −1 −3 0 0−2 8 −3 −3 −1 −2 −1 −2 −1 −2 −2 2 −3 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 2 −31 0 −3 −2 −1 −3 −1 3 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 −2 2 0 −3 −2 −1 −2−1 1 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 −1 −3 −1 0 −1 −3 −2 −2 M −1 −1 −2−3 −1 0 −2 −3 −2 1 2 −1 5 0 −2 −1 −1 −1 −1 1 F −2 −3 −3 −3 −2 −3 −3 −3−1 0 0 −3 0 6 −4 −2 −2 1 3 −1 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2−4 7 −1 −1 −4 −3 −2 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 1 −3 −2 −2T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 −2 −2 0 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 2 −3 Y −2 −2 −2 −3 −2 −1 −2 −32 −1 −1 −2 −1 3 −3 −2 −2 2 7 −1 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1−2 −2 0 −3 −1 4.

As used herein, “sequence identity” refers to a relationship between twoor more polynucleotide sequences or between two or more polypeptidesequences. When a position in one sequence is occupied by the samenucleic acid base or amino acid residue in the corresponding position ofthe comparator sequence, the sequences are said to be “identical” atthat position. The percentage sequence identity is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof identical positions. The number of identical positions is thendivided by the total number of positions in the comparison window andmultiplied by 100 to yield the percentage of sequence identity.Percentage of sequence identity is determined by comparing two optimallyaligned sequences over a comparison window. The comparison window forpolynucleotide sequences can be, for instance, at least 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,300, 400, 500, 600, 700, 800, 900 or 1000 or more nucleic acids inlength. The comparison window for polypeptide sequences can be, forinstance, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 300 or more amino acids in length. Inorder to optimally align sequences for comparison, the portion of apolynucleotide or polypeptide sequence in the comparison window cancomprise additions or deletions termed gaps while the reference sequenceis kept constant. An optimal alignment is that alignment which, evenwith gaps, produces the greatest possible number of “identical”positions between the reference and comparator sequences. Percentage“sequence identity” between two sequences can be determined using theversion of the program “BLAST 2 Sequences” which was available from theNational Center for Biotechnology Information as of Sep. 1, 2004, whichprogram incorporates the programs BLASTN (for nucleotide sequencecomparison) and BLASTP (for polypeptide sequence comparison), whichprograms are based on the algorithm of Karlin and Altschul (Proc. Natl.Acad. Sci. USA 90(12):5873-5877, 1993). When utilizing “BLAST 2Sequences,” parameters that were default parameters as of Sep. 1, 2004,can be used for word size (3), open gap penalty (11), extension gappenalty (1), gap dropoff (50), expect value (10) and any other requiredparameter including but not limited to matrix option. Two nucleotide oramino acid sequences are considered to have “substantially similarsequence identity” or “substantial sequence identity” if the twosequences have at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity relative to each other.

The term “monoclonal antibody” (Mab) refers to an antibody, orantigen-binding portion thereof, that is derived from a single copy orclone, including for example any eukaryotic, prokaryotic, or phageclone, and not the method by which it is produced. Preferably, amonoclonal antibody of the invention exists in a homogeneous orsubstantially homogeneous population.

A “humanized” antibody molecule refers to a form of non-human (forexample, murine) antibody molecules, or antigen-binding portion thereof,that are chimeric immunoglobulins, immunoglobulin chains, or fragmentsthereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-bindingsub-sequences of antibodies) that contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies may be humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity.

“Human antibody or fully human antibody” refers to an antibody molecule,or antigen-binding portion thereof, derived from transgenic micecarrying human antibody genes or from human cells.

The term “chimeric antibody” is intended to refer to an antibodymolecule, or antigen-binding portion thereof, in which the variableregion sequences are derived from one species and the constant regionsequences are derived from another species, such as an antibody moleculein which the variable region sequences are derived from a mouse antibodyand the constant region sequences are derived from a human antibody.

“Antibody-drug conjugate” and “immunoconjugate” refer to an antibodymolecule, or antigen-binding portion thereof, including antibodyderivatives that binds to C-KIT, which is conjugated to cytotoxic,cytostatic and/or therapeutic agents.

Antibody molecules of the invention, or antigen-binding portion thereof,can be produced using techniques well known in the art, for examplerecombinant technologies, phage display technologies, synthetictechnologies or combinations of such technologies or other technologiesreadily known in the art.

The term “isolated molecule” (where the molecule is, for example, apolypeptide, a polynucleotide, or an antibody) is a molecule that byvirtue of its origin or source of derivation (1) is not associated withnaturally associated components that accompany it in its native state,(2) is substantially free of other molecules from the same species (3)is expressed by a cell from a different species, or (4) does not occurin nature. Thus, a molecule that is chemically synthesized, or expressedin a cellular system different from the cell from which it naturallyoriginates, will be “isolated” from its naturally associated components.A molecule also may be rendered substantially free of naturallyassociated components by isolation, using purification techniques wellknown in the art. Molecule purity or homogeneity may be assayed by anumber of means well known in the art. For example, the purity of apolypeptide sample may be assayed using polyacrylamide gelelectrophoresis and staining of the gel to visualize the polypeptideusing techniques well known in the art. For certain purposes, higherresolution may be provided by using HPLC or other means well known inthe art for purification.

The term “epitope” refers to that portion of a molecule capable of beingrecognized by and bound by an antibody molecule, or antigen-bindingportion thereof, at one or more of the antibody molecule'santigen-binding regions. Epitopes can consist of defined regions ofprimary secondary or tertiary protein structure and includescombinations of secondary structural units or structural domains of thetarget recognised by the antigen-binding regions of the antibody, orantigen-binding portion thereof. Epitopes can likewise consist of adefined chemically active surface grouping of molecules such as aminoacids or sugar side chains and have specific three-dimensionalstructural characteristics as well as specific charge characteristics.The term “antigenic epitope” as used herein, is defined as a portion ofa polypeptide to which an antibody molecule can specifically bind asdetermined by any method well known in the art, for example, byconventional immunoassays, antibody competitive binding assays or byx-ray crystallography or related structural determination methods (forexample NMR).

The term “binding affinity” or “KD” refers to the dissociation rate of aparticular antigen-antibody interaction. The KD is the ratio of the rateof dissociation, also called the “off-rate (k_(off))”, to theassociation rate, or “on-rate (k_(on))”. Thus, K_(D) equalsk_(off)/k_(on) and is expressed as a molar concentration (M). It followsthat the smaller the K_(D), the stronger the affinity of binding.Therefore, a K_(D) of 1 μM indicates weak binding affinity compared to aK_(D) of 1 nM. KD values for antibodies can be determined using methodswell established in the art. One method for determining the KD of anantibody is by using surface plasmon resonance (SPR), typically using abiosensor system such as a BIACORE® system.

The term “potency” is a measurement of biological activity and may bedesignated as IC₅₀, or effective concentration of an antibody orantibody drug conjugate to the antigen C-KIT to inhibit 50% of activitymeasured in a C-KIT activity assay as described herein.

The phrase “effective amount” or “therapeutically effective amount” asused herein refers to an amount necessary (at dosages and for periods oftime and for the means of administration) to achieve the desiredtherapeutic result. An effective amount is at least the minimal amount,but less than a toxic amount, of an active agent which is necessary toimpart therapeutic benefit to a subject.

The term “inhibit” or “neutralize” as used herein with respect tobioactivity of an antibody molecule of the invention means the abilityof the antibody to substantially antagonize, prohibit, prevent,restrain, slow, disrupt, eliminate, stop, reduce or reverse for exampleprogression or severity of that which is being inhibited including, butnot limited to, a biological activity or binding interaction of theantibody molecule to C-KIT.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

As used herein, “vector” means a construct, which is capable ofdelivering, and, preferably, expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

The term “treating”, as used herein, unless otherwise indicated, meansreversing, alleviating, inhibiting the progress of, delaying theprogression of, delaying the onset of, or preventing the disorder orcondition to which such term applies, or one or more symptoms of suchdisorder or condition. The term “treatment”, as used herein, unlessotherwise indicated, refers to the act of treating as defined above. Theterm “treating” also includes adjuvant and neoadjuvant treatment of asubject. For the avoidance of doubt, reference herein to “treatment”includes reference to curative, palliative and prophylactic treatment.For the avoidance of doubt, references herein to “treatment” alsoinclude references to curative, palliative and prophylactic treatment.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent specification, including definitions, will control. Throughoutthis specification and claims, the word “comprise,” or variations suchas “comprises” or “comprising” will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers. Unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Any example(s) following the term “e.g.” or “forexample” is not meant to be exhaustive or limiting.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art.

Particular non-limiting embodiments of the present invention will now bedescribed with reference to accompanying drawings.

Example 1. Generation of Optimized Anti-C-KIT Therapeutic AntibodiesIntroduction

In this example, we successfully generated a panel of antagonistic,optimized anti-C-KIT antibodies. These anti-C-KIT antibodies were wellexpressed, biophysically stable, highly soluble and of maximized aminoacid sequence identity to preferred human germlines.

Materials and Methods

IgG Cloning, Transient Expression, Purification

Antibody v-domain encoding DNA sequences were cloned viarestriction-ligation cloning into separate IgG heavy and light-chainexpression cassettes in separate plasmid vectors.

Antibodies were expressed in two human IgG1 formats: IgG1 andIgG1null-IgG1 with the lower hinge mutations L234A/L235A/G237A, whichminimise Fcγ receptor-driven effector functions. IgGs were expressed inHEK-293expi cells after transient transfection with endotoxin-free IgGexpression plasmid preparations, per manufacturer's protocols. IgGs werepurified using a single-step protocol: Conditioned media were loaded(neat) onto a 1 ml ProA sepharose column, pre-equilibrated in PBS pH7.4.The column was washed with 5 column volumes of PBS pH7.4, before theprotein was eluted with 100 mM glycine, pH 2.7 and subjected to dialysisin PBS pH 7.4 using 30 kDa cutoff dialysis membrane.

IgG Titration Binding ELISAs

To coat Greiner Bio-One High bind ELISA plates, target proteins werediluted to 1 μg/ml in carbonate buffer and added at 100 μl per well, at4° C., o/n. Coated plates were washed 3× with PBS pH7.4, blocked with 1%BSA in PBS (380 μl/well) for 1 hr at RT, then washed 3× with PBS-Tween20 (PBST). C-KIT antibodies (100 μl/well; diluted in PBST) were thenadded and then incubated 1 hr at RT. Plates were then washed 3× withPBST and goat anti-human kappa chain-HRP added (100 μl/well) at RT, for1 hr. Plates were then washed 3× with PBST and twice with PBS before theaddition of 100 μl TMB per well. Reactions were stopped by adding 100 μl2M H₂SO₄/well and OD was read on a plate reader at 450 nm.

Anti-C-KIT antibodies were tested for polyreactivity by ELISA. Purified,recombinant, target and non-target antigens were coated in 96-well Nuncmaxisorp plates at 100 ng per well in carbonate buffer, at 4° C.overnight. Plates were then washed 3× with PBS, blocked with 1% BSA inPBS, then washed 3× with PBS-Tween20. A dilution series of primaryantibodies was then applied, plates were washed 3× with PBS-Tween20followed by application of goat anti-human kappa chain-HRP 1:4,000secondary antibody. Wells were then washed 3× with PBS-Tween20 and 2×with PBS, 100 μl TMB peroxidase substrate was added per well, thereaction was stopped by adding 100 μl 2M H₂SO₄ and absorbances were readat 450 nm. IgG binding analysis via ELISA on negatively chargedbiomolecular surfaces were performed as previously described (seeMouquet et al., 2010, Nature 467: 591-595).

C-KIT Library Generation and Selection

The C-KIT scFv repertoire was assembled by mass oligonucleotidesynthesis and PCR. The amplified scFv repertoire was then cloned viarestriction-ligation into a phagemid vector, transformed into E. coliTG-1 cells, and the phage repertoire rescued essentially as previouslydescribed in detail (Finlay et al., 2011, Methods Mol Biol 681:383-401).

Phage selections were performed by coating streptavidin magneticmicrobeads with C-KIT-Fc protein (either human or cyno), washing thebeads thrice with PBS and resuspending in PBS pH7.4 plus 5% skim milkprotein (MPBS). These beads were coated at 200 nM target protein inround 1 of selection, followed by 100, 50 and 10 nM in subsequentrounds.

HTRF Binding Competition Assay

A competition homogeneous time resolved fluorescence (HTRF) assay wasestablished to examine epitope competition against h37M IgG by graftedand library-derived clones. The purified h37M IgG1 was labelled withterbium using a labelling kit (CisBio) per the manufacturersinstructions. The final reaction mix contained biotinylated humanC-KIT-Fc, SA-XL665 (CisBio), terbium-labelled parental h37M, andcompetitor IgG of interest, prepared as described above, in a totalreaction volume of 20 μl in 1× assay buffer [50 mM sodium phosphate, pH7.5, 400 mM potassium fluoride, and 0.1% BSA (w/v)]. Reagents were addedsequentially into 384-well low-volume black plates (Nunc). Reactionsproceeded for 1 h at room temperature, and plates were subsequently readon a plate reader with excitation at 340 nm and two emission readings at615 nm (measuring input donor fluorescence from h37M-terbium) and 665 nm(measuring output acceptor fluorescence from SAXL665). Readings wereexpressed as 665 nm/615 nm ratios.

Antibody v-Domain T Cell Epitope Content: In Silico Analyses

In silico technologies (Abzena, Ltd.), which are based on identifyingthe location of T cell epitopes in therapeutic antibodies and proteins,were used for assessing potential immunogenicity in antibody v-domains.iTope™ was used to analyse the VL and VH sequences of key leads forpeptides with promiscuous high affinity binding to human MHC class II.Promiscuous high affinity MHC class II binding peptides are thought tocorrelate with the presence of T cell epitopes that are high riskindicators for clinical immunogenicity of drug proteins. The iTope™software predicts favourable interactions between amino acid side chainsof a peptide and specific binding pockets (in particular pocketpositions; p1, p4, p6, p7 and p9) within the open-ended binding groovesof 34 human MHC class II alleles. These alleles represent the mostcommon HLA-DR alleles found world-wide with no weighting attributed tothose found most prevalently in any particular ethnic population. Twentyof the alleles contain the ‘open’ p1 configuration and 14 contain the‘closed’ configuration where glycine at position 83 is replaced by avaline. The location of key binding residues is achieved by the insilico generation of 9 mer peptides that overlap by eight amino acidsspanning the test protein sequence. This process successfullydiscriminates with high accuracy between peptides that either bind or donot bind MHC class II molecules.

In addition, the sequences were analysed using TCED™ (T Cell EpitopeDatabase™) search for matches to T cell epitopes previously identifiedby in vitro human T cell epitope mapping analyses of other proteinsequences. The TCED™ is used to search any test sequence against a large(>10,000 peptides) database of peptides derived from unrelated proteinand antibody sequences.

BIACORE® Analyses of IgG Affinity for Human Fc Receptors

Interaction affinities for IgGs were determined by surface plasmonresonance using a BIACORE® T200 instrument. For most analyses,His6-tagged FcγRI, FcγRIIa (167R and 167H variants), FcγRIIb, FcγRIIIa(176F and 176 V variants), and FcγRIIIb receptors (all Sino Biological)were captured on a CM5 sensor chip coated with an anti-HIS antibody bystandard amine coupling. Receptor-specific formats of analyses were thenapplied, as below.

FcγRI is a high-affinity receptor for IgG1 monomers, so 1:1 kineticanalysis was performed under the following conditions: ‘single cycle’analysis using flow rate 30 μl/min, receptor protein loaded to ˜30 RU at10 μl/min (diluted 0.25 μg/ml in HBS-P+), 5 point three-fold dilution ofpurified antibodies titrated 0.411 nM to 33.33 nM applied with anassociation time of 200 s, dissociation time of 300 s. Regeneration with2× injections glycine pH 1.5 and analysis using 1:1 fit.

The interactions between monomeric IgG and FcγRII and FcγRIII receptorsare relatively low affinity interactions, so ‘steady state’ affinityanalyses were performed under the following conditions: flow rate 30μl/min, receptor protein loaded to ˜60 RU at 10 μl/min (diluted 0.25μg/ml in HBS-P+), 5 point three-fold dilution series of purifiedantibodies titrated between 33 nM and 24000 nM applied with anassociation time of 30 s, dissociation time of 25 s. Regeneration with2× injections glycine pH 1.5 and analysis using steady state affinitycalculation.

The interactions between monomeric IgG and FcRn are relatively lowaffinity and pH-sensitive interactions, so ‘steady state’ affinityanalyses were performed under the following conditions: A CM5 chip wasdirectly coupled with hFcRn in sodium acetate pH 5.5 using standardamine chemistry. Running buffer was PBS 0.05% P20+150 mM NaCl (pH 6.0 orpH 7.4), flow rate 30 μl/min, 5 point three-fold dilutions of purifiedantibodies from 3000 nM to 37.0 nM applied with an association time of18 s, dissociation time 100 s. Regeneration with 0.1 M Tris pH 8.0 andanalysis using steady state affinity calculation.

Antibody v-Domain Specificity Testing: Human Receptor Array Analyses

Human cell membrane receptor proteome arrays were performed atRetrogenix Ltd. Primary screens: IgG1-h37M and IgG1-MH1 antibodies werescreened for binding against fixed HEK293 cells/slides expressing 5528human plasma membrane and cell surface-tethered secreted proteinsindividually (n=2 slides per slide set). All transfection efficienciesexceeded the minimum threshold. Antibody binding was detected usingAF647 fluorescent secondary anti-human IgG1 antibody. Primary hits(duplicate spots) were identified by analysing fluorescence (AF647 andZsGreen1) on ImageQuant. Vectors encoding all hits were sequenced toconfirm their correct identities. Confirmation/specificity screens:Vectors encoding all hits, plus control vectors encoding MS4A1 (CD20),EGFR and other proteins, were spotted in duplicate on new slides, andused to reverse transfect human HEK293 cells as before. All transfectionefficiencies exceeded the minimum threshold. Identical fixed slides weretreated with 0.5 and 2 μg/ml of each test antibody, 1 μg/ml of thenegative control antibody, 1 μg/ml Rituximab biosimilar (positivecontrol), or no test molecule (secondary only; negative control) (n=1slide per treatment). Slides were analysed as above.

Analyses of Internalisation and Cell-Killing Potential in Armed AntibodyFormats

CHO cells stably expressing human or cyno c-KIT, grown in Ham's F12media containing 20% Fetal Bovine Serum, 1 mM L-Glutamine and 1 μg/mlG418 were seeded into 384-well black clear bottomed tissue culturetreated assay plates (500 cells in 30 μl per well) and incubatedovernight in a CO₂ incubator at 37° C. Purified antibodies were seriallydiluted in media then an equal volume of 120 nM Fab-ZAP added (AdvancedTargeting Systems, IT-51). The antibody/Fab-ZAP mixtures were incubatedfor 30 minutes at 37° C. before being added (10 μl per well) to the cellassay plates. The cells were incubated for 72 hours in CO₂ incubator at37° C. On each plate, background controls (media only) and Fab-ZAPcontrol wells (cells incubated with FAB-ZAP reagent but no c-KITantibody) were included for the purposes of data normalisation. At theend of the 72-hour incubation, cell viability was determined using theCellTiter-Glo® Cell Viability assay (Promega G7571) according to themanufacturer's instructions. The relative luminescent signal (RLU) foreach well was measured using the BMG FLUOstar Omega plate reader. Thedata was blank corrected by subtraction of the RLU signal of the mediaonly wells and expressed as a % of the blank corrected signal of theFab-ZAP control wells.

TF-1 cells in RPMI media containing 10% Fetal Bovine Serum, 2 mML-Glutamine, 10 mM HEPES, 4.5 g/l D-Glucose, penicillin, streptomycinand 2 ng/ml GM-CSF were seeded into 96-well black clear bottomed tissueculture treated assay plates (2500 cells in 75 μl per well). Purifiedantibodies were serially diluted in media then an equal volume of 40 nMFab-ZAP added. The antibody/Fab-ZAP mixtures were incubated for 30minutes at 37° C. before being added (25 μl per well) to the cell assayplates. The cells were incubated for 72 hours in CO₂ incubator at 37° C.On each plate, background controls (media only) and Fab-ZAP controlwells (cells incubated with FAB-ZAP reagent but no c-KIT antibody) wereincluded for the purposes of data normalisation. At the end of the72-hour incubation, cell viability was determined using theCellTiter-Glo® Cell Viability assay (Promega G7571) according to themanufacturer's instructions. The relative luminescent signal (RLU) foreach well was measured using the BMG FLUOstar Omega plate reader. Thedata was blank corrected by subtraction of the RLU signal of the mediaonly wells and expressed as a % of the blank corrected signal of theFab-ZAP control wells.

Analyses of Antibody Potency in C-KIT Receptor Activity Neutralisation

TF-1 cells in RPMI media containing 10% Fetal Bovine Serum, 2 mML-Glutamine, 10 mM HEPES, 4.5 g/l D-Glucose, penicillin, streptomycinand 2 ng/ml GM-CSF were seeded into 96-well clear bottomed tissueculture treated assay plates. Purified antibodies were serially dilutedin media then added to the cell assay plates and incubated for 60minutes at 37° C. Recombinant h-SCF was then added at 50 ng/ml and thecells were incubated for 72 hours in CO₂ incubator at 37° C. On eachplate, background controls (media only) were included for the purposesof data normalisation. At the end of the 72-hour incubation, cellviability was determined using the resazurin fluorescence viabilityassay according to the manufacturer's instructions.

Results and Discussion

CDR Grafting onto Preferred Human Germline v-Genes

The CDRs of an antagonistic murine anti-C-KIT IgG 37M (37M; seeWO2014018625A1 and Table 2) were initially introduced to human germlineimmunoglobulin v-domain framework sequence scaffolds using CDR grafting.To bias our engineering efforts towards final lead therapeutic IgGcompounds with optimal drug-like properties, we chose to graft the CDRsof the parental antibody onto “preferred” germline scaffolds IGHV1-46and IGKV1-16, which are known to have good solubility, high physicalstability and are used at high frequency in the expressed human antibodyrepertoire.

Those scaffolds and grafted CDR definitions are outlined in Table 2. Theheavy and light chain sequences for chimeric anti-C-KIT antibody m37Mand humanized h37M are also shown in Table 2. While this process of CDRgrafting is well known, it is still problematic to predict whether agiven set of human v-domain sequences will act as suitable acceptorframeworks for non-human CDR grafting. The use of unsuitable frameworkscan lead to the loss of target binding function, protein stabilityissues or even impaired expression of the final IgG. Indeed, theinclusion of multiple murine residues in the framework regions of h37Mare indicative that full target binding affinity could not be maintainedin the direct CDR graft into germline frameworks. The IGHV1-46/IGKV1-16graft was therefore taken forward as the template for CDR mutagenesisand selection of improved clones.

Library Generation and Screening

The CDR-grafted IGKV1-16/IGHV1-46 v-domain sequences were combined intoa VL-VH scFv format and a mutagenesis library cassette was generated bymass oligonucleotide synthesis and assembly. The final scFv library wasligated into a phage display vector and transformed into E. coli viaelectroporation to generate 1.0×10⁹ independent clones. Library buildquality was verified by sequencing 96 clones. This sequencing datashowed that the positions encoding either the murine or human germlineresidue at each position of variance had been effectively sampled at afrequency of approximately 50%. Libraries were rescued using helperphage M13 and selections performed on biotinylated human and cynomolgusmonkey C-KIT-Fc proteins in multiple separate branches.

Post-selection screening (as shown in FIG. 1) and DNA sequencingrevealed the presence of 219 unique, human and mouse C-KIT-binding scFvclones that retained epitope binding competition with h37M IgG1 andcontained significantly increased human content within the CDRs, whilethe framework sequences remained fully germline. Amongst these 219clones, germ-lining mutations were observed in all CDRs (Table 3). Leadclones were ranked based on the level of CDR germ-lining versus ELISAand HTRF signals for both human and cyno C-KIT-Fc (FIG. 1). Thev-domains of the 13 top clones from this ranking were then sub-clonedinto IgG expression vectors for further testing as below (Table 4).

While germ-lining mutations were observed in all CDRs for the leadclones derived directly from library selections, it remained possiblethat sequence analyses might allow further clones to be designed to havemaximal humanization. The 219 sequence-unique hits with binding signalsagainst human and mouse protein were therefore used to analyse theretention frequency for murine amino acids in the CDRs of thisfunctionally characterized population. Positional amino acid retentionfrequency was expressed as a percentage found in the V_(H) and V_(L)domains (FIG. 2A, 2B). Murine residues with RF<75% were regarded aspositions that are possibly not essential to the target-binding paratopeand are likely to be open to germ-lining, in a series of combinatorialdesigns.

Fifteen designs containing principally those murine residues withRF>75%, in a number of combinations, were designated “MH1-MH15”(MH=Maximally Humanized). As the LCDR3 of h37M contained a potentialdeamidation site (‘NS’ at positions 3 and 4 of the CDR), multiple ‘MH’clones also sampled possible substitutions such as N/NS/T/E and S/A/N/D,which might remove this development risk motif while maintainingacceptable target binding function. The sole non-human germline residuefound in the LCDR3 (‘R’ at position 8 in the CDR) was also sampled forthe tolerance of homologous substitutions R/K/H, to establish ifstability or immunogenicity-enhancing mutations might be identified.Another six designer clones “TTP1-6” (‘TTP’=Total TheoreticallyPossible) were also created that combined the most humanized CDRsobserved in the high-functioning population of scFv sequences, plusdeamidation motif-disrupting mutations (Table 4). The MH and TTP cloneswere generated by gene synthesis (along with the 13 library-derivedclones outlined above, positive controls h37M and m37M, and negativecontrol non-C-KIT-reactive isotype v-domains), then cloned intoexpression vectors for production as human IgG1. All IgGs were readilyexpressed and purified from transient transfections of HEK-293 cells.

Lead IgG Specificity and Potency Characteristics

The purified IgGs described above were then tested for binding to humanand cyno C-KIT-Fc in direct titration ELISA format (FIG. 3). Thisanalysis demonstrated that while all library derived clones and MH1-3,5, 10, 11 and 12 designer clones retained binding affinity for human andcyno C-KIT that was comparable to the h37M IgG1, all of the other MH andTTP clones showed reduced binding to both orthologues (FIG. 3).

As direct ELISA binding signal is influenced by avidity and does notprove the maintenance a specific epitope, all IgGs were then examined ina solution-phase HTRF competition assay (FIG. 4). All library-derivedand multiple designer IgGs exhibited full concentration-dependentinhibition of h37M binding to human (FIG. 4A) and cyno (FIG. 4C, D)c-KIT, with key leads showing highly similar IC50 values to thatobserved for unlabelled h37M IgG (Table 5). This demonstratedmaintenance of a shared epitope and binding affinity in these clones.All TTP clones, plus MH clones 4, 6, 7 and 9 were found not to fullyinhibit h37M binding to either human (FIG. 4B) or cyno (FIG. 4E) C-KIT.

Flow Cytometric Analyses of Lead IgG Binding Specificity at the CellMembrane

Antibodies to C-KIT were analysed for concentration-dependent binding atthe cell surface via flow cytometry. CHO-K1 cells were stablytransfected with either human or cyno C-KIT full-length cDNAs.Anti-C-KIT IgGs and an isotype control IgG1 were then all tested in IgG1format, over a concentration range of 100-0.02 μg/ml for binding tohuman (FIG. 5A, 5B), cyno (FIG. 5C, 5D) or wild type control (‘wt’, i.e.untransfected) CHO-K1 (FIG. 5E, 5F). All IgGs other than the isotypecontrol showed concentration-dependent binding to human and cyno C-KIT+cells, with a maximum MFI in each case being >50-fold higher thanobserved background signals for binding to untransfected CHO-K1.Anti-C-KIT antibodies exhibited no measurable background binding onuntransfected CHO-K1 cells, comparable to the Isotype control IgG1 (FIG.5E, 5F).

Analyses of Designer IgGs Based on the Lead Clone MH5

As described above, clone MH5 had proven to have highly specific bindingto human and cyno C-KIT, low off-target binding potential, reduceddeamidation potential in the CDRs, fully germline framework regions andmultiple human germlining mutations in the CDRs. As a first-generationdesigner clone, however, the MH5 sequence had been targeted for improvedhuman germline content and development qualities in the light chain, butnot the heavy chain. The VH domain still retained a number ofnon-germline (mouse-derived) residues in the CDRs that were suggested tobe potentially modifiable by the data found in FIGS. 2A and 2B andobserved functional mutant sequences outlined in Table 3. In an attemptto sample further mutations in the VH that might improve expression,stability, immunogenicity or functional characteristics, 36 ‘secondgeneration’ mutants were generated as outlined in Table 6. These cloneswere expressed and purified in IgG1 format and examined for targetbinding by ELISA on human and cyno C-KIT orthologs (FIGS. 6A and 6B,respectively) and neutralisation of h37M/C-KIT interaction for bothorthologs by HTRF. In this phase, it was found that strong ELISA bindingsignals for several clones suggested multiple mutations could betolerated in the CDRs of the majority of the IgGs, but these changescould not always be combined without some changes in potency. In theHTRF analyses (FIG. 7A, 7B) it was found that key second generationleads MH5.1, 5.2, 5.22, 5.23, 5.24, 5.33 and 5.34, in particular, werecapable of fully neutralising h37M/C-KIT interaction, albeit withreduced potency by IC50 calculation (Table 7).

Finally, the second-generation clones MH5.1, 5.2, 5.22, 5.23, 5.24, 5.33and 5.34 were examined for their binding to human (FIG. 8A), cyno (FIG.8B) or wild type control (‘wt’, i.e. untransfected) CHO-K1 (FIG. 8C)cells by flow cytometry. These analyses confirmed that each of these 7prioritised second-generation clones still exhibit strong,concentration-dependent binding to human or cyno KIT+ CHO-K1 cells thatis near identical to the signals generated by h37M and MH5, but alsoshow no observable binding to untransfected CHO-K1.

Lead IgG Analyses in ‘Developability’ ELISA Assays

To ensure that high-affinity lead clones had not suffered from loss oftarget specificity during the mutation and reselection process; clonesE-C2, E-C7, MH1, MH5, MH5.22 and h37M were tested for binding to a panelof 14 purified human proteins from the immunoglobulin superfamily (FIG.9A-F). All six IgGs exhibited strong binding signals at 1 μg/ml toC-KIT-Fc (human OD450 nm>1.8, cyno>2.0), and no detectable binding(OD450 nm<0.1) against any other protein, including the closely-relatedmurine and rat C-KIT-Fc proteins, which demonstrated that target bindingspecificity had been maintained in these clones.

It is known in the art that the binding of IgGs intended for therapeuticuse to several indicative biological substrates is an indicator of highrisk for poor performance in patients due to poor bioavailability andshort in vivo half-life. Three such biological substrates are Insulin,dsDNA and ssDNA. These three substrates were therefore used to coatELISA plates and examine the binding of the IgG1null versions of theoptimised lead antibodies. Binding signals for these human IgG-basedantibodies was compared to ‘positive control’ human IgG antibodies thathave been found to have polyreactivity and poor performance, whichstopped their progress in clinical trials (Bococizumab and Briakinumabhuman IgG1 analogues). For a negative control human IgG1 antibody, anIgG1 Ustekinumab analogue was used as it reacts with the sametherapeutic target as Briakinumab, but has longer pK and wassuccessfully approved as a therapeutic product. In the ELISA analysesshown in FIGS. 10A, B and C, the positive control antibodies exhibitedthe expected strong reactivity to all 3 substrates, while the negativecontrol Ustekinumab showed low reactivity. Importantly, all of the IgG1lead clones tested showed binding≤ the negative control against all 3substrates. This finding underlined the maintenance of highly specific,target-driven binding in the optimised clones E-C2, E-C7, MH1, MH5 andMH5.22.

Antibody v-Domain T Cell Epitope Analyses

In silico technologies (Abzena, Ltd.), which are based on identifyingthe location of T cell epitopes in therapeutic antibodies and proteins,were used for assessing the immunogenicity of both the h37M and leadantibody v-domains. Analysis of the v-domain sequences was performedwith overlapping 9 mer peptides (with each overlapping the last peptideby 8 residues) which were tested against each of the 34 MHC class IIallotypes. Each 9 mer was scored based on the potential ‘fit’ andinteractions with the MHC class II molecules. The peptide scorescalculated by the software lie between 0 and 1. Peptides that produced ahigh mean binding score (>0.55 in the iTope™ scoring function) werehighlighted and, if >50% of the MHC class II binding peptides (i.e. 17out of 34 alleles) had a high binding affinity (score>0.6), suchpeptides were defined as ‘high affinity’ MHC class II binding peptideswhich are considered a high risk for containing CD4+ T cell epitopes.Low affinity MHC class II binding peptides bind a high number of alleles(>50%) with a binding score>0.55 (but without a majority>0.6). Furtheranalysis of the sequences was performed using the TCED™. The sequenceswere used to interrogate the TCED™ by BLAST search in order to identifyany high sequence homology between peptides (T cell epitopes) fromunrelated proteins/antibodies that stimulated T cell responses inprevious in vitro T cell epitope mapping studies performed at AbzenaLtd.

Peptides were grouped into four classes: High Affinity Foreign(‘HAF’—high immunogenicity risk), Low Affinity Foreign (‘LAF’—lowerimmunogenicity risk), TCED+(previously identified epitope in TCED™database), and Germline Epitope (‘GE’—human germline peptide sequencewith high MHC Class II binding affinity). Germline Epitope 9 merpeptides are unlikely to have immunogenic potential due to T celltolerance, as validated by previous studies with a wide range ofgermline peptides. Importantly, such germline v-domain epitopes (aidedfurther by similar sequences in the human antibody constant regions)also compete for MHC Class II occupancy at the membrane of antigenpresenting cells, reducing the risk of foreign peptide presentationbeing sufficient to achieve the ‘activation threshold’ required for Tcell stimulation. High GE content is therefore a beneficial quality inclinical development of an antibody therapeutic.

As shown in FIG. 11 and Table 8, key lead v-domains exhibitedsignificant beneficial changes in peptide epitope content in comparisonto h37M. As the v-domain engineering process undertaken here hadsuccessfully selected for antibodies that maintained anti-KIT potencywithout the need for the murine residues included in the frameworks ofh37M (Table 2), multiple HAF and LAF epitopes found in the frameworks ofboth the heavy and light chain v-domains of h37M (FIG. 11A) were absentin all library-derived and designer leads (Table 8). GE epitope contentwas also found to be significantly increased (from 4 to ≥10 in allleads), particularly in the VH regions of lead clones (FIG. 11B-11H),and TCED+ epitopes were reduced or eliminated in all leads (Table 8).Importantly, however, multiple foreign epitopes were also eliminated bygermlining mutations found in the CDRs of lead clones. For example, aTCED+ and HAF peptide ‘VTITCKASQ’ (SEQ ID NO: 64) found in the LCDR-1 ofh37M was eliminated in all lead clones by the mutation K>R at position6, converting this sequence to the light chain GE ‘VTITCRASQ’ (SEQ IDNO: 65; FIG. 11B-11H). Similarly, for clones MH1 (FIG. 11E) and MH5.22(FIG. 11G), the HAF peptides ‘LIYSASSLQ’ (SEQ ID NO: 66) and ‘IYSASSLQS’(SEQ ID NO: 67) were both converted to GE sequences by mutation of theLCDR-2 sequence to the fully germline sequence ‘AASSLQS’ (SEQ ID NO:24). The stabilising mutations found in the LCDR-3 of clones MH5 andMH5.22 (Tables 4 and 6), which are mutations away from the IGKV1-16germline, were not found to generate any epitopes.

In the VH region of h37M, the peptide sequence ‘YYINWVRQA’ (SEQ ID NO:68; spanning the HCDR-1 and FW2) was found to be both a TCED+ and HAF.The HCDR-1 germlining mutation I>M at position 3, found in all leadsother than MH5.22 (Table 6), eliminated this risk and converted thepeptide sequence into a GE. For clone MH5.22 however, the mutation Y>Win the HCDR-3 compensated by eliminating two LAF peptide sequences (FIG.11G, Table 8). The above findings allowed the generation of the fullysequence-optimised clone MH5-DI (by changing the LCDR-2 sequence of MH5from SASSLQS (SEQ ID NO: 17) to AASSLQS (SEQ ID NO: 24)), which hadminimised chemical stability and immunogenicity risk characteristics inits primary sequence (Table 8, FIG. 11H).

BIACORE® Analyses of Affinity of IgG Variants for Human Fc Receptors

Antibodies targeting the KIT receptor have been shown in human clinicalstudies and in vitro human immunological analyses to have a high risk ofinjection reactions in patients due to Fc receptor cross-linking duringKIT engagement on mast cells (L'Italien et al., Clin Cancer Res24(14):3465-3474, 2018). In order to examine receptor-engagementpotential of Fc-engineered variants of the anti-KIT antibodies, h37M wasexpressed in IgG1 and IgG1-3M (L234A/L235A/G237A) formats, while cloneMH1 was expressed in the novel IgG1-4M format (L234A/L235A/G237A/P331S).Each purified IgG1 variant was then examined for binding affinity to allhuman Fc receptors via surface plasmon resonance analyses. Theseanalyses demonstrated that both the isotype control human IgG1 andh37M-IgG1 exhibited strong binding affinity for all human Fcγ receptors(Table 9) and normal affinity for FcRn at pH 6.0 but not pH 7.4 (Table10). In contrast, while h37M-IgG1-3M and MH1 IgG1-4M also demonstratednormal affinity for FcRn at pH 6.0 but not pH 7.4 (Table 10), they bothexhibited no measurable binding affinity for any human Fcγ receptorsother than the inhibitory receptor FcγRIIb (Table 9). The lowest signalson FcγRIIb were observed for the MH1 IgG-4M (FIG. 12). As demonstratedby the isotype control human IgG4 antibody, use of this isotype reducesthe affinity of the antibody to most Fcγ receptors, but is notsufficient to fully ablate activating receptor interaction (Table 9).

Lack of binding to activating FcγRI, FcγRIIa, FcγRIIIa and FcγRIIIb, butmaintained binding to inhibitory receptor FcγRIIb and recycling receptorFcRn is an ideal combination of characteristics for an anti-KITtherapeutic antibody in either standard IgG form or as an armed antibody(used in antibody drug conjugate form, or in targeting anotherimmune-activating mechanism such as CD3 ligation, CD16A ligation or CD47blockade), as this potentially maximises the half-life of the moleculein circulation and simultaneously minimises the risk of mast cellactivation-related toxicity in man.

Human Extracellular Proteome Array Analyses of Antibody Specificity

The exquisite specificity of antibody target recognition is one of theprimary reasons they are chosen as drug candidates. This specificity isnot guaranteed however, and impaired specificity after in vitroengineering of antibodies is a risk. To examine this possibility in theantibodies generated here, in vitro technologies (Retrogenix, Ltd.),which are based on using high-density arrays of cells expressing 5528unique human plasma membrane and cell surface-tethered secretedproteins, were used to screen for off-target binding specificities inIgG1-h37M and IgG1-MH1. This receptor array binding screen identifiedthat both antibodies exhibited strong binding to membrane-expressedC-KIT, but also had 3 potential off-target binding specificities: MMP7,CRIM1 and F13A1. Analysis of these interactions on re-array slidesshowed that these interactions were in fact artefactual and weremediated by the secondary antibody (FIG. 13A). For IgG1-h37M andIgG1-MH1 (FIG. 13B, 13C), they therefore showed extreme specificity,both binding only to the 2 known isoforms of C-KIT: accession numbersNM_000222.2 (canonical isoform; SEQ ID NO:209) and NM_001093772 (shortisoform; SEQ ID NO:210). Repeat analyses of clones E-C2, E-C7, F-05 andMH5 also exhibited highly specific binding to both C-KIT isoforms.

Cell-Killing Potential in Armed Antibody Formats and Comparison withC-KIT/SCF Neutralisation Potency

A key characteristic of antibody drug conjugates is the ability tointernalise into cells expressing the antibody's cognate target.Internalisation of anti-c-KIT antibodies was assessed using a goatpolyclonal heavy and light chain specific Fab conjugated to saporintoxin. The Fab-ZAP reagent binds to the human IgG1 and if that IgG1binds to a protein on a cell surface and is internalised, then theFab-ZAP reagent is also internalised into endosomes. Once in theendosome, the saporin toxin is released from the complex and inactivatesribosomes, eventually resulting in cell death. This method thereforeallows direct comparison of the potential potency of IgG1 antibodies asantibody drug conjugates. In these internalisation experiments, allantibodies h37M, MH1 and MH5-DI in IgG1-3M form drove highly potentinternalisation and killing of CHO cells expressing human C-KIT (FIG.14A), cyno C-KIT (FIG. 14B), and the TF-1 human erythroleukaemia line(FIG. 14C), while isotype control human IgG1 did not drive anyinternalisation or killing of any of the 3 cell types. Importantly, all3 clones also generated highly similar potencies, as measured in IC50,with all 3 IC50 values being within ˜2-fold on KIT+ CHO cells and within˜4-fold on TF-1 cells (Table 11). These highly similar values forpotency in cell-killing experiments were expected, as clones MH1 and MH5exhibited fully overlapping curves with h37M (and thereforeindistinguishable affinities for C-KIT) in high-sensitivity,solution-phase HTRF epitope competition for binding to human C-KIT (FIG.15A) and cyno C-KIT (FIG. 15B), as above.

As the erythroleukaemia line TF-1 constitutively expresses C-KIT, it isdriven to proliferate when the natural ligand for C-KIT, SCF, is addedto culture media. This provided an ideal experimental context in whichto examine the potency of the anti-C-KIT IgGs in the neutralisation ofC-KIT/SCF signalling. TF-1 cells were cultured in the presence of SCFand multiple antibodies tested for their ability to inhibit cellularproliferation. This analysis demonstrated that all antibodies testedwere capable of inhibiting cellular proliferation being driven by SCF,but that h37M was significantly more potent than any other clone (FIG.16). Indeed, when IC50 values were generated, it was found that onlyh37M exhibited potency in the pM range (0.11 nM), while MH1 and MH5-DIwere 26.5-fold and 243.6-fold less potent, respectively (Table 11).

This potency differential in C-KIT/SCF inhibition versus internalisationand toxin delivery is a significant unexpected benefit for clones suchas MH1 and MH5-DI. C-KIT is highly expressed on haematopoietic stemcells in the bone marrow, so anti-C-KIT antibody drug conjugates for thetreatment of cancer exhibit improved therapeutic index when theyefficiently deliver toxins in the pM concentration range but areincapable of blocking C-KIT/SCF signalling when dosed at such lowconcentrations. This leads to improved tumor targeting, but reduced bonemarrow toxicity (L′Italien et al., Clin Cancer Res 24(14):3465-3474,2018). Reduced potency in C-KIT/SCF signalling inhibition, but retentionof high affinity binding to the C-KIT ectodomain may also prove to bebeneficial for improving the therapeutic index of other forms of armedanti-C-KIT antibody, for example in immune targeting via CD3 ligation,CD16A ligation, or CD47 blockade. Clones described here, such as MH1 andMH5-DI in IgG1-4M format may therefore have the ideal set of improvedcharacteristics over h37M-IgG1 for development as armed anti-C-KITantibodies: low immunogenicity, high affinity/high specificity targetingof C-KIT, high potency delivery of toxins via internalisation, lowerpotency C-KIT/SCF signalling inhibition and no interaction with humanFcγ receptors.

Although the present invention has been described with reference topreferred or exemplary embodiments, those skilled in the art willrecognize that various modifications and variations to the same can beaccomplished without departing from the spirit and scope of the presentinvention and that such modifications are clearly contemplated herein.No limitation with respect to the specific embodiments disclosed hereinand set forth in the appended claims is intended nor should any beinferred.

All documents cited herein are incorporated by reference in theirentirety.

TABLE 1 Amino acid sequences murine anti-C-KIT CDRs as defined here(“Unified” scheme) in comparison to alternative definitions. SchemeHCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Unified GYTFTDYYINIARIYPGSGNTYYNEKFKG GVYYFDY KASQNVRTNVA SASYRYS QQYNSYPRT (SEQ ID(SEQ ID NO: 5) (SEQ ID (SEQ ID NO: 10) (SEQ ID NO: (SEQ ID NO: NO: 4)NO: 6) 11) 12) Kabat DYYIN RIYPGSGNTYYNEKFKG GVYYFDY KASQNVRTNVA SASYRYSQQYNSYPRT (SEQ ID (SEQ ID NO: 73) (SEQ ID (SEQ ID NO: 10) (SEQ ID NO:(SEQ ID NO: NO: 69) NO: 6) 11) 12) Chothia GYTFTDY YPGSGN GVYYFDYKASQNVRTNVA SASYRYS QQYNSYPRT (SEQ ID (SEQ ID NO: 74) (SEQ ID(SEQ ID NO: 10) (SEQ ID NO: (SEQ ID NO: NO: 70) NO: 6) 11) 12) IMGTGYTFTDYY IYPGSGNT ARGVYYFDY QNVRTN SAS QQYNSYPRT (SEQ ID (SEQ ID NO: 75)(SEQ ID (SEQ ID NO: 82) (SEQ ID NO: NO: 71) NO: 79) 12) AHo GYTFTDYYINIYPGSGNTYYNEKFKG GVYYFD ASQNVRTN SASYRYS YNSYPR (SEQ ID (SEQ ID NO: 76)(SEQ ID (SEQ ID NO: 83) (SEQ ID NO: (SEQ ID NO: NO: 4) NO: 80) 11) 86)AbM GYTFTDYYIN RIYPGSGNTY GVYYFDY KASQNVRTNVA SASYRYS QQYNSYPRT (SEQ ID(SEQ ID NO: 77) (SEQ ID (SEQ ID NO: 10) (SEQ ID NO: (SEQ ID NO: NO: 4)NO: 6) 11) 12) Contact TDYYIN IARIYPGSGNTY ARGVYYFD VRTNVAWY ALIYSASYRYQQYNSYPR (SEQ ID (SEQ ID NO: 78) (SEQ ID (SEQ ID NO: 84) (SEQ ID NO:(SEQ ID NO: NO: 72) NO: 81) 85) 207)

TABLE 2 Amino acid sequence of 37M murine anti-C-KIT v-domains (m37M)and human germline CDR grafts (h37M). V DOMAIN Human germline¹Amino acid sequence² m37M-VH n/a QVQLKQSGAELVRPGASVKLSCKAS GYTFTDYYINWVKQRPGQGLEW IARIYPGSGNTYYNEKFKG KATLTAEKSSSTAYMQLSSLTSEDSAVYFCARGVYYFDY WGQGTTLTVSS (SEQ ID NO: 87) h37M-VH IGHV1-46³QVQLVQSGAEVKKPGASVK

SCKAS GYTFTDYYIN WVRQAPG

GLEW IARIYPGSGNTYYNEKFKG R

T

T

D

STST

YM

LSSLRSEDTAVY

CAR GVYYFDY WGQGTTVTVSS (SEQ ID NO: 88) VH graft IGHV1-46⁴QVQLVQSGAEVKKPGASVKVSCKAS GYTFTDYYIN WVRQAPGQGLEW IARIYPGSGNTYYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GVYYFDY WGQGTLVTVSS (SEQ ID NO: 89)m37M-VL n/a DIVMTQSQKFMSTSVGDRVSVTC KASQNVRTNVA WYQQKPGQSPKALIY SASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLADYFC QQYNSYPRT FGGGTKLEIKR (SEQ ID NO: 90)h37M-VL IGKV1-16³ DI

MTQSPSSLSASVGDRVTITC KASQNVRTNVA W

QQKPGKAPK

LIY SASYRYS GVP

RF

GSGSGTDFTLTISSLQPEDFA

YFC QQYNSYPRT FGGGTKVEIK (SEQ ID NO: 91) VL graft IGKV1-16⁴DIQMTQSPSSLSASVGDRVTITC RASQGVRTNVA WFQQKPGKAPKSLIY SASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYNSYPRT FGGGTKVEIK (SEQ ID NO: 92)¹Human germline definitions used for grafting, based on IMGT system.²CDR residues are in bold and underlined. As noted above, the“Unified” CDR definitions used in this manuscript are an expandeddefinition in comparison to the classical Kabat definition. Eachsequence above shows the framework regions (FRs) and the CDRs in thefollowing order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. ³h37M domains containseveral murine residues in the framework regions, shown here italicized.⁴Grafts are fully germline in the framework regions, used as thetemplate for CDR mutant library construction.

TABLE 3Amino acid sequences of unique CDRs from 219 unique anti-C-KIT v-domains.LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 RASQGIRNNLA AASSLQS QQYASYPLTGYTFTDYYMH IAIIYPGSGNTYYAQKFQG GVYYFDA (SEQ ID NO: (SEQ ID (SEQ ID(SEQ ID NO: (SEQ ID NO: 126) (SEQ ID NO: 93) NO: 24) NO: 119) 123) 151)RASQGIRNNVA AASSLYS QQYASYPRT GYTFTDYYMN IARINPGSGNTSYAQKFQG GVYYFDD(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID NO: (SEQ ID NO: 127) (SEQ ID NO: 94)NO: 111) NO: 53) 13) 152) RASQGIRNYVA AASSRQS QQYNSYPLT GYTFTSYYINIARINPGSGNTYYAQKFQG GVYYFDE (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID NO:(SEQ ID NO: 128) (SEQ ID NO: 95) NO: 23) NO: 120) 124) 21) RASQGIRTNLAAASSRYS QQYQSYPLT GYTFTSYYMN IARIYPGSGNTSYAQKFQG GVYYFDF (SEQ ID NO:(SEQ ID (SEQ ID (SEQ ID NO: (SEQ ID NO: 129) (SEQ ID NO: 16) NO: 112)NO: 121) 125) 153) RASQGIRTNVA AASYLQS QQYQSYPRT IARIYPGSGNTYYAQKFQGGVYYFDG (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID NO: 206) (SEQ ID NO: 19)NO: 50) NO: 122) 154) RASQGISNYLA AASYLYS IARIYPSSGNTSYAQKFQG GVYYFDH(SEQ ID NO: (SEQ ID (SEQ ID NO: 130) (SEQ ID NO: 96) NO: 113) 155)RASQGISNYVA AASYRQS IARIYPSSGNTYYAQKFQG GVYYFDI (SEQ ID NO: (SEQ ID(SEQ ID NO: 131) (SEQ ID NO: 97) NO: 44) 156) RASQGISTNLA AASYRYSIGIIYPGSGNTYYAQKFQG GVYYFDK (SEQ ID NO: (SEQ ID (SEQ ID NO: 132)(SEQ ID NO: 98) NO: 114) 157) RASQGISTNVA SASSLQS IGRINPGSGNTSYAQKFQGGVYYFDL (SEQ ID NO: (SEQ ID (SEQ ID NO: 133) (SEQ ID NO: 99) NO: 17)205) RASQGISTYLA SASSLYS IGRINPGSGNTYYAQKFQG GVYYFDM (SEQ ID NO: (SEQ ID(SEQ ID NO: 134) (SEQ ID NO: 100) NO: 115) 158) RASQGISTYVA SASSRQSIGRIYPGSGNTSYAQKFQG GVYYFDN (SEQ ID NO: (SEQ ID (SEQ ID NO: 135)(SEQ ID NO: 101) NO: 52) 159) RASQGVRNNLA SASSRYS IGRIYPGSGNTYYAQKFQGGVYYFDP (SEQ ID (SEQ ID (SEQ ID NO: 45) (SEQ ID NO: NO: 43) NO: 116)160) RASQGVRNNVA SASYLQS IGRIYPSSGNTSYAQKFQG GVYYFDQ (SEQ ID NO: (SEQ ID(SEQ ID NO: 136) (SEQ ID NO: 47) NO: 117) 161) RASQGVRNYVA SASYLYSIGRIYPSSGNTYYAQKFQG GVYYFDR (SEQ ID NO: (SEQ ID (SEQ ID NO: 137)(SEQ ID NO: 102) NO: 118) 162) RASQGVRTNLA SASYRQS MAIIYPGSGNTYYAQKFQGGVYYFDS (SEQ ID NO: (SEQ ID (SEQ ID NO: 138) (SEQ ID NO: 22) NO: 20) 46)RASQGVRTNVA YASSLQS MARINPGSGNTSYAQKFQG GVYYFDT (SEQ ID NO: (SEQ ID(SEQ ID NO: 139) (SEQ ID NO: 39) NO: 48) 51) RASQGVSNNLAMARINPGSGNTYYAQKFQG GVYYFDV (SEQ ID NO: (SEQ ID NO: 140) (SEQ ID NO:103) 163) RASQGVSNNVA MARIYPGSGNTSYAQKFQG GVYYFDW (SEQ ID NO:(SEQ ID NO: 141) (SEQ ID NO: 104) 164) RASQGVSNYLA MARIYPGSGNTYYAQKFQGGVYYADY (SEQ ID NO: (SEQ ID NO: 142) (SEQ ID NO: 105) 165) RASQGVSNYVAMARIYPSSGNTSYAQKFQG GVYYGDY (SEQ ID NO: SEQ ID NO: 143) (SEQ ID NO: 106)166) RASQGVSTNLA MARIYPSSGNTYYAQKFQG GVYYHDY (SEQ ID NO:(SEQ ID NO: 144) (SEQ ID NO: 107) 167) RASQGVSTNVA MGIIYPGSGNTYYAQKFQGGVYYQDY (SEQ ID NO: (SEQ ID NO: 145) (SEQ ID NO: 108) 41) RASQGVSTYLAMGRINPGSGNTSYAQKFQG GVYYLDY (SEQ ID NO: (SEQ ID NO: 146) (SEQ ID NO:109) 18) RASQGVSTYVA MGRINPGSGNTYYAQKFQG GVYYKDY (SEQ ID NO:(SEQ ID NO: 147) (SEQ ID NO: 110) 168) MGRIYPGSGNTSYAQKFQG GVYYNDY(SEQ ID NO: 148) (SEQ ID NO: 169) MGRIYPGSGNTYYAQKFQG GVYYMDY(SEQ ID NO: 14) (SEQ ID NO: 170) MGRIYPSSGNTSYAQKFQG GVYYYDY(SEQ ID NO: 149) (SEQ ID NO: 15) MGRIYPSSGNTYYAQKFQG GVYYWDY(SEQ ID NO: 150) (SEQ ID NO: 171) GVYAFDY (SEQ ID NO: 172) GVYQFDY(SEQ ID NO: 173) GVYHFDY (SEQ ID NO: 49) GVYWFDY (SEQ ID NO: 174)GVYNFDY (SEQ ID NO: 175) GVYEFDY (SEQ ID NO: 42) GVYTFDY (SEQ ID NO:176) GVYDFDY (SEQ ID NO: 177) GVWYFDY (SEQ ID NO: 40)

TABLE 4Amino acid sequences of CDRs of unique, library-derived and designer,human/cyno cross-reactive anti-C-KIT antibodies. Clone Name LCDR1 LCDR2LCDR3 HCDR1 HCDR2 HCDR3 E-E10 RASQGVRTNVA SASSLQS QQYNSYPRT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVWYFDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 39) NO: 17) NO: 12) NO: 13) NO: 40) F-F2RASQGVRTNVA AASSRQS QQYNSYPRT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYQDY(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 39) NO: 23)NO: 12) NO: 13) NO: 41) C-B12 RASQGVRNNLA AASYRQS QQYNSYPRT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYEFDY (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID NO: 43) NO: 44) NO: 12) NO: 13) NO: 42) C-A7RASQGVRNNVA YASSLQS QQYNSYPRT GYTFTDYYMN IGRIYPGSGNTYYAQKFQG GVYYFDS(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 45) (SEQ ID 47) NO: 48)NO: 12) NO: 13) NO: 46) C-A5 RASQGVRNNVA AASYLQS QQYNSYPRT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYHFDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 47) NO: 50) NO: 12) NO: 13) NO: 49) D-A10RASQGVRTNLA AASSRQS QQYNSYPRT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYFDT(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 22) NO: 23)NO: 12) NO: 13) NO: 51) E-C7 RASQGIRTNLA SASSLQS QQYNSYPRT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYYYDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 16) NO: 17) NO: 12) NO: 13) NO: 15) D-D5RASQGVRTNLA SASSLQS QQYNSYPRT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYYDY(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 22) NO: 17)NO: 12) NO: 13) NO: 15) E-C2 RASQGIRTNVA SASYRQS QQYNSYPRT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYYLDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 19) NO: 20) NO: 12) NO: 13) NO: 18) F-B11RASQGIRTNLA AASYRQS QQYNSYPRT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYLDY(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 16) NO: 44)NO: 12) NO: 13) NO: 18) D-D9 RASQGVRTNVA SASSLQS QQYNSYPRT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYYLDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 39) NO: 17) NO: 12) NO: 13) NO: 18) E-G7RASQGVRTNVA SASSRQS QQYNSYPRT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYFDE(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 39) NO: 52)NO: 12) NO: 13) NO: 21) F-C5 RASQGVRTNLA AASSRQS QQYNSYPRT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYYFDE (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 22) NO: 23) NO: 12) NO: 13) NO: 21) MH1RASQGIRTNLA AASSLQS QQYNSYPRT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYYDY(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 16) NO: 24)NO: 12) NO: 13) NO: 15) MH2 RASQGIRTNLA SASSLQS QQYASYPRT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYYYDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 16) NO: 17) NO: 53) NO: 13) NO: 15) MH3RASQGIRTNLA SASSLQS QQYNAYPRT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYYDY(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 16) NO: 17)NO: 54) NO: 13) NO: 15) MH4 RASQGIRTNLA SASSLQS QQYNDYPRT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYYYDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 16) NO: 17) NO: 55) NO: 13) NO: 15) MH5RASQGIRTNLA SASSLQS QQYANYPRT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYYDY(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 16) NO: 17)NO: 25) NO: 13) NO: 15) MH6 RASQGIRTNLA SASSLQS QQYNSYPKT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYYYDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 16) NO: 17) NO: 56) NO: 13) NO: 15) MH7RASQGIRTNLA SASSLQS QQYNSYPHT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYYDY(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 16) NO: 17)NO: 57) NO: 13) NO: 15) MH8 RASQGIRTNLA SASSLQS QQYSSYPRT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYYYDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 16) NO: 17) NO: 58) NO: 13) NO: 15) MH9RASQGIRTNLA SASSLQS QQYESYPRT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYYDY(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 16) NO: 17)NO: 59) NO: 13) NO: 15) MH10 RASQGIRTNLA SASSLQS QQYTSYPRT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYYYDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 16) NO: 17) NO: 60) NO: 13) NO: 15) MH11RASQGIRTNLA AASSLQS QQYNAYPRT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYYDY(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 16) NO: 24)NO: 54) NO: 13) NO: 15) MH12 RASQGVRTNLA AASSLQS QQYNSYPRT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYYYDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 22) NO: 24) NO: 12) NO: 13) NO: 15) MH13RASQGVRTNLA AASSLQS QQYNAYPRT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYYDY(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 22) NO: 24)NO: 54) NO: 13) NO: 15) MH14 RASQGVRTNLA AASSLQS QQYNSYPRT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYYLDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 22) NO: 24) NO: 12) NO: 13) NO: 18) MH15RASQGVRTNLA AASSLQS QQYNAYPRT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYLDY(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 22) NO: 24)NO: 54) NO: 13) NO: 18) TTP1 RASQGIRTNLA AASSLQS QQYNSYPKT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYYLDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 16) NO: 24) NO: 56) NO: 13) NO: 18) TTP2RASQGIRTNLA AASSLQS QQYNSYPHT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYYDY(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 16) NO: 24)NO: 57) NO: 13) NO: 15) TTP3 RASQGVRTNLA SASSLQS QQYNAYPKT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYYLDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 22) NO: 17) NO: 178) NO: 13) NO: 18) TTP4RASQGVRTNLA SASSLQS QQYNAYPHT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYYDY(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 22) NO: 17)NO: 179) NO: 13) NO: 15) TTP5 RASQGVRTNLA AASSLQS QQYNAYPKT GYTFTDYYMNMGRIYPGSGNTYYAQKFQG GVYYLDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 14) (SEQ ID 22) NO: 24) NO: 178) NO: 13) NO: 18) TTP6RASQGVRTNLA AASSLQS QQYNAYPHT GYTFTDYYMN MGRIYPGSGNTYYAQKFQG GVYYYDY(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 14) (SEQ ID 22) NO: 24)NO: 179) NO: 13) NO: 15)

TABLE 5 HTRF epitope competition IC50 values for key library derived anddesigner lead anti-C-KIT antibodies in IgG1 format. Human Cyno CloneName IC50 (μg/ml) IC50 (μg/ml) C-KIT-h37M 0.025 0.035 C-KIT-D-A10 0.0270.059 C-KIT-E-C7 0.028 0.051 C-KIT-E-E10 0.031 0.097 C-KIT-F-C5 0.0310.079 C-KIT-C-A7 0.037 0.081 C-KIT-F-F2 0.046 0.121 C-KIT-MH11 0.0510.101 C-KIT-MHS 0.063 0.036 C-KIT-C-B12 0.066 0.166

TABLE 6Amino acid sequences of CDRs of second generation, designer, human/cynocross-reactive anti-C-KIT antibodies. Clone Name LCDR1 LCDR2 LCDR3 HCDR1HCDR2 HCDR3 MH5.1 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTNYYMNMGRIYPGTGNTYYAQKFQG GVWYYDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 61) (SEQ ID 16) NO: 24) NO: 25) NO: 181) NO: 27) MH5.2RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDFYMN MGRIYPGTGNTYYAQKFQG GVWYYDY(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 61) (SEQ ID 16) NO: 24)NO: 25) NO: 180) NO: 27) MH5.3 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDHYMNMGRIYPGTGNTYYAQKFQG GVWYYDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 61) (SEQ ID 16) NO: 24) NO: 25) NO: 182) NO: 27) MH5.4RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDHYMN MGRIYPGTGNTYYAQKFQG GVWYFDS(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 61) (SEQ ID 16) NO: 24)NO: 25) NO: 182) NO: 62) MH5.5 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDHYMNMGRIYPGTGNTYYAQKFQG GVWYFDT (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 61) (SEQ ID 16) NO: 24) NO: 25) NO: 182) NO: 63) MH5.6RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDHYMN MGRIYPGTGNTYYAQKFQG GVWYFDE(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 61) (SEQ ID 16) NO: 24)NO: 25) NO: 182) NO: 184) MH5.7 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDHYMNMGRIYPGAGNTYYAQKFQG GVWYYDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 183) (SEQ ID 16) NO: 24) NO: 25) NO: 182) NO: 27) MH5.8RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDHYMN MGRIYPGAGNTYYAQKFQG GVWYFDS(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 183) (SEQ ID 16) NO: 24)NO: 25) NO: 182) NO: 62) MH5.9 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDHYMNMGRIYPGAGNTYYAQKFQG GVWYFDT (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 183) (SEQ ID 16) NO: 24) NO: 25) NO: 182) NO: 63) MH5.10RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDHYMN MGRIYPGAGNTYYAQKFQG GVWYFDE(SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 183) (SEQ ID 16) NO: 24)NO: 25) NO: 182) NO: 184) MH5.11 RASQGIRTNLA AASSLQS QQYANYPRTGYTFTDHYMN MGRIYPASGNTYYAQKFQG GVWYYDY (SEQ ID NO: (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 26) (SEQ ID 16) NO: 24) NO: 25) NO: 182) NO: 27)MH5.12 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDHYMN MGRIYPASGNTYYAQKFQGGVWYFDS (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 26) (SEQ ID 16)NO: 24) NO: 25) NO: 182) NO: 62) MH5.13 RASQGIRTNLA AASSLQS QQYANYPRTGYTFTDHYMN MGRIYPASGNTYYAQKFQG GVWYFDT (SEQ ID NO: (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 26) (SEQ ID 16) NO: 24) NO: 25) NO: 182) NO: 63)MH5.14 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDHYMN MGRIYPASGNTYYAQKFQGGVWYFDE (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 26) (SEQ ID 16)NO: 24) NO: 25) NO: 182) NO: 184) MH5.15 RASQGIRTNLA AASSLQS QQYANYPRTGYTFTDFYMN MGRIYPGTGNTYYAQKFQG GVWYFDS (SEQ ID NO: (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 61) (SEQ ID 16) NO: 24) NO: 25) NO: 180) NO: 62)MH5.16 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDFYMN MGRIYPGTGNTYYAQKFQGGVWYFDT (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 61) (SEQ ID 16)NO: 24) NO: 25) NO: 180) NO: 63) MH5.17 RASQGIRTNLA AASSLQS QQYANYPRTGYTFTDFYMN MGRIYPGTGNTYYAQKFQG GVWYFDE (SEQ ID NO: (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 61) (SEQ ID 16) NO: 24) NO: 25) NO: 180) NO: 184)MH5.18 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDFYMN MGRIYPGAGNTYYAQKFQGGVWYYDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 183) (SEQ ID 16)NO: 24) NO: 25) NO: 180) NO: 27) MH5.19 RASQGIRTNLA AASSLQS QQYANYPRTGYTFTDFYMN MGRIYPGAGNTYYAQKFQG GVWYFDS (SEQ ID NO: (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 183) (SEQ ID 16) NO: 24) NO: 25) NO: 180) NO: 62)MH5.20 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDFYMN MGRIYPGAGNTYYAQKFQGGVWYFDT (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 183) (SEQ ID 16)NO: 24) NO: 25) NO: 180) NO: 63) MH5.21 RASQGIRTNLA AASSLQS QQYANYPRTGYTFTDFYMN MGRIYPGAGNTYYAQKFQG GVWYFDE (SEQ ID NO: (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 183) (SEQ ID 16) NO: 24) NO: 25) NO: 180) NO: 184)MH5.22 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDFYMN MGRIYPASGNTYYAQKFQGGVWYYDY (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 26) (SEQ ID 16)NO: 24) NO: 25) NO: 180) NO: 27) MH5.23 RASQGIRTNLA AASSLQS QQYANYPRTGYTFTDFYMN MGRIYPASGNTYYAQKFQG GVWYFDS (SEQ ID NO: (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 26) (SEQ ID 16) NO: 24) NO: 25) NO: 180) NO: 62)MH5.24 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTDFYMN MGRIYPASGNTYYAQKFQGGVWYFDT (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 26) (SEQ ID 16)NO: 24) NO: 25) NO: 180) NO: 63) MH5.25 RASQGIRTNLA AASSLQS QQYANYPRTGYTFTDFYMN MGRIYPASGNTYYAQKFQG GVWYFDE (SEQ ID NO: (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 26) (SEQ ID 16) NO: 24) NO: 25) NO: 180) NO: 184)MH5.26 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTNYYMN MGRIYPGTGNTYYAQKFQGGVWYFDS (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 61) (SEQ ID 16)NO: 24) NO: 25) NO: 181) NO: 62) MH5.27 RASQGIRTNLA AASSLQS QQYANYPRTGYTFTNYYMN MGRIYPGTGNTYYAQKFQG GVWYFDT (SEQ ID NO: (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 61) (SEQ ID 16) NO: 24) NO: 25) NO: 181) NO: 63)MH5.28 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTNYYMN MGRIYPGTGNTYYAQKFQGGVWYFDE (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 61) (SEQ ID 16)NO: 24) NO: 25) NO: 181) NO: 184) MH5.29 RASQGIRTNLA AASSLQS QQYANYPRTGYTFTNYYMN MGRIYPGAGNTYYAQKFQG GVWYYDY (SEQ ID NO: (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 183) (SEQ ID 16) NO: 24) NO: 25) NO: 181) NO: 27)MH5.30 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTNYYMN MGRIYPGAGNTYYAQKFQGGVWYFDS (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 183) (SEQ ID 16)NO: 24) NO: 25) NO: 181) NO: 62) MH5.31 RASQGIRTNLA AASSLQS QQYANYPRTGYTFTNYYMN MGRIYPGAGNTYYAQKFQG GVWYFDT (SEQ ID NO: (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 183) (SEQ ID 16) NO: 24) NO: 25) NO: 181) NO: 63)MH5.32 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTNYYMN MGRIYPGAGNTYYAQKFQGGVWYFDE (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 183) (SEQ ID 16)NO: 24) NO: 25) NO: 181) NO: 184) MH5.33 RASQGIRTNLA AASSLQS QQYANYPRTGYTFTNYYMN MGRIYPASGNTYYAQKFQG GVWYYDY (SEQ ID NO: (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 26) (SEQ ID 16) NO: 24) NO: 25) NO: 181) NO: 27)MH5.34 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTNYYMN MGRIYPASGNTYYAQKFQGGVWYFDS (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 26) (SEQ ID 16)NO: 24) NO: 25) NO: 181) NO: 62) MH5.35 RASQGIRTNLA AASSLQS QQYANYPRTGYTFTNYYMN MGRIYPASGNTYYAQKFQG GVWYFDT (SEQ ID NO: (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 26) (SEQ ID 16) NO: 24) NO: 25) NO: 181) NO: 63)MH5.36 RASQGIRTNLA AASSLQS QQYANYPRT GYTFTNYYMN MGRIYPASGNTYYAQKFQGGVWYFDE (SEQ ID NO: (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 26) (SEQ ID 16)NO: 24) NO: 25) NO: 181) NO: 184)

TABLE 7 HTRF epitope competition IC50 values for second generationdesigner anti-C-KIT antibodies in IgG1 format. Human Cyno Clone NameIC50 (μg/ml) IC50 (μg/ml) C-KIT-h37M 0.016 0.036 C-KIT-MH5 0.067 0.058C-KIT-MH5.1 0.684 0.633 C-KIT-MH5.2 0.272 0.254 C-KIT-MH5.22 0.134 0.164C-KIT-MH5.23 2.024 3.256 C-KIT-MH5.24 2.201 2.743 C-KIT-MH5.34 0.8390.532 C-KIT-MH5.35 2.047 0.785

TABLE 8 Human T cell epitope content in v-domains predicted by iTOPE ™and TCED ™. Germline High Affinity Low Affinity Clone Name epitopesForeign Foreign TCED+ C-KIT-h37M 4 7 6 3 C-KIT-E-C7 10 3 4 0 C-KIT-F-C510 3 4 1 C-KIT-E-C2 10 2 4 0 C-KIT-MH1 12 1 4 0 C-KIT-MH5 10 3 4 0C-KIT-MH5.22 11 3 1 1 C-KIT-MH5-DI 12 1 2 0

TABLE 9 BIACORE ® analyses of affinity of IgG variants for human Fcreceptors. Receptor Antibody KD (M) Chi2 FcG RI Isotype IgG1 3.44E−090.0289 FcG RI Isotype IgG4 9.25E−09 0.016 FcG RI h37M-IgG1 1.06E−090.573 FcG RI h37M-IgG1-3M NS NS FcG RI MH1-IgG1-4M NS NS FcG RIIa (167R)Isotype IgG1 3.71E−06 3.57 FcG RIIa (167R) Isotype IgG4 Low Low FcG RIIa(167R) h37M-IgG1 4.47E−07 23.7 FcG RIIa (167R) h37M-IgG1-3M NS NS FcGRIIa (167R) MH1-IgG1-4M NS NS FcG RIIa (167H) Isotype IgG1 6.14E−06 2.09FcG RIIa (167H) Isotype IgG4 Low Low FcG RIIa (167H) h37M-IgG1 5.12E−074.16 FcG RIIa (167H) h37M-IgG1-3M NS NS FcG RIIa (167H) MH1-IgG1-4M NSNS FcG RIIb Isotype IgG1 1.72E−05 1.55 FcG RIIb Isotype IgG4 2.31E−050.431 FcG RIIb h37M-IgG1 1.44E−06 2.58 FcG RIIb h37M-IgG1-3M Low Low FcGRIIb MH1-IgG1-4M Low Low FcG RIIIa (176F) Isotype IgG1 3.53E−06 0.0343FcG RIIIa (176F) Isotype IgG4 NS NS FcG RIIIa (176F) h37M-IgG1 8.20E−080.205 FcG RIIIa (176F) h37M-IgG1-3M NS NS FcG RIIIa (176F) MH1-IgG1-4MNS NS FcG RIIIa (176V) Isotype IgG1 1.40E−06 0.448 FcG RIIIa (176V)Isotype IgG4 Low Low FcG RIIIa (176V) h37M-IgG1 4.11E−08 3.36 FcG RIIIa(176V) h37M-IgG1-3M NS NS FcG RIIIa (176V) MH1-IgG1-4M NS NS FcG RIIIbIsotype IgG1  9.9E−06 1.85 FcG RIIIb Isotype IgG4 NS NS FcG RIIIbh37M-IgG1 2.49E−07 5.6 FcG RIIIb h37M-IgG1-3M NS NS FcG RIIIbMH1-IgG1-4M NS NS NS = No Signal observed at any concentration of IgGLow = Signal observed at high IgG concentrations

TABLE 10 BIACORE ® analyses of affinity of IgG variants for human FcRn.pH Antibody KD (M) Chi2 6 Isotype IgG1 1.18E−06 0.756 7.4 Isotype IgG1NS NS 6 Isotype IgG4  2.6E−06 0.213 7.4 Isotype IgG4 NS NS 6 h37M-IgG11.24E−06 8.75 7.4 h37M-IgG1 NS NS 6 h37M-IgG1-3M 1.32E−06 6.58 7.4h37M-IgG1-3M NS NS 6 MH1-IgG1-4M 1.59E−06 3 7.4 MH1-IgG1-4M NS NS NS =No Signal observed at any concentration of IgG

TABLE 11 Potency values for anti-C-KIT antibodies in IgG format incell-based assays. TF-1 TF-1 Human C-KIT Cyno C-KIT proliferation FabZAPCHO FabZAP CHO FabZAP Clone Name IC50 (nM) IC50 (pM) IC50 (pM) IC50 (pM)C-KIT-h37M 0.11 12.76 5.7 7.823 C-KIT-MH1 2.86 51.09 8.7 13.96C-KIT-MH5-DI 26.31 53.68 12.6 13.54

TABLE 12  Examples of antibody variable region amino acid sequences.Antibody MH1 heavy chain variable (VH) regionQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRIYPGSGNTYYAQKFQGRVTMTRDTSISTVYMELSSLRSEDTAVYYCARGVYYYDYWGQGTLVTVSS (SEQ ID NO: 185)Antibody MH1 VL light chain variable (VL) regionDIQMTQSPSSLSASVGDRVTITCRASQGIRTNLAWFQQKPGKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 186)Antibody MH5-DI heavy chain variable (VH) regionQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRIYPGSGNTYYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVYYYDYWGQGTLVTVSS (SEQ ID NO: 187)Antibody MH5-DI light chain variable (VL) regionDIQMTQSPSSLSASVGDRVTITCRASQGIRTNLAWFQQKPGKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYANYPRTFGGGTKVEIK (SEQ ID NO: 188)Antibody MH5 heavy chain variable (VH) regionQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRIYPGSGNTYYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVYYYDYWGQGTLVTVSS (SEQ ID NO: 189)Antibody MH5 light chain variable (VL) regionDIQMTQSPSSLSASVGDRVTITCRASQGIRTNLAWFQQKPGKAPKSLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYANYPRTFGGGTKVEIK (SEQ ID NO: 190)Antibody EC7 heavy chain variable (VH) regionQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRIYPGSGNTYYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVYYYDYWGQGTLVTVSS (SEQ ID NO: 191)Antibody EC7 light chain variable (VL) regionDIQMTQSPSSLSASVGDRVTITCRASQGIRTNLAWFQQKPGKAPKSLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 192)Antibody EC2 heavy chain variable (VH) regionQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRIYPGSGNTYYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVYYLDYWGQGTLVTVSS (SEQ ID NO: 193)Antibody EC2 light chain variable (VL) regionDIQMTQSPSSLSASVGDRVTITCRASQGIRTNVAWLQQKPGKAPKSLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 194)Antibody F-C5 heavy chain variable (VH) regionQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGRIYPGSGNTYYAQKFQGRVTMTRDTSISTVYMELSSLRSEDTAVYYCARGVYYFDEWGQGTLVTVSS (SEQ ID NO: 195)Antibody F-C5 light chain variable (VL) regionDIQMTQSPSSLSASVGDRVTITCRASQGVRTNLAWFQQKPGKAPKSLIYAASSRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 196)

TABLE 13 Examples of antibody Fc region amino acid sequences.Human IgG1 wild type ASTKGPSVFPLAPSSKSISGGTAALGCLVKDYFPEPVTVSWNSGALISGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 197)Human IgG1-3M ASTKGPSVFPLAPSSKSISGGTAALGCLVKDYFPEPVTVSWNSGALISGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 198)Human IgG1-4M ASTKGPSVFPLAPSSKSISGGTAALGCLVKDYFPEPVTVSWNSGALISGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 199)Human IgG1 wild type “REEM” allotypeASTKGPSVFPLAPSSKSISGGTAALGCLVKDYFPEPVTVSWNSGALISGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 200)Human IgG1-3M “REEM” allotypeASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 201)Human IgG1-4M “REEM” allotypeASTKGPSVFPLAPSSKSISGGTAALGCLVKDYFPEPVTVSWNSGALISGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 202)

TABLE 14 Examples of human C-KIT amino acid and nucleotide sequences.Human C-KIT proteinMRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAATAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELAIDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAHESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV(SEQ ID NO: 208) Human C-KIT protein, transcript variant 1TCTGGGGGCTCGGCTTTGCCGCGCTCGCTGCACTTGGGCGAGAGCTGGAACGTGGACCAGAGCTCGGATCCCATCGCAGCTACCGCGATGAGAGGCGCTCGCGGCGCCTGGGATTTTCTCTGCGTTCTGCTCCTACTGCTTCGCGTCCAGACAGGCTCTTCTCAACCATCTGTGAGTCCAGGGGAACCGTCTCCACCATCCATCCATCCAGGAAAATCAGACTTAATAGTCCGCGTGGGCGACGAGATTAGGCTGTTATGCACTGATCCGGGCTTTGTCAAATGGACTTTTGAGATCCTGGATGAAACGAATGAGAATAAGCAGAATGAATGGATCACGGAAAAGGCAGAAGCCACCAACACCGGCAAATACACGTGCACCAACAAACACGGCTTAAGCAATTCCATTTATGTGTTTGTTAGAGATCCTGCCAAGCTTTTCCTTGTTGACCGCTCCTTGTATGGGAAAGAAGACAACGACACGCTGGTCCGCTGTCCTCTCACAGACCCAGAAGTGACCAATTATTCCCTCAAGGGGTGCCAGGGGAAGCCTCTTCCCAAGGACTTGAGGTTTATTCCTGACCCCAAGGCGGGCATCATGATCAAAAGTGTGAAACGCGCCTACCATCGGCTCTGTCTGCATTGTTCTGTGGACCAGGAGGGCAAGTCAGTGCTGTCGGAAAAATTCATCCTGAAAGTGAGGCCAGCCTTCAAAGCTGTGCCTGTTGTGTCTGTGTCCAAAGCAAGCTATCTTCTTAGGGAAGGGGAAGAATTCACAGTGACGTGCACAATAAAAGATGTGTCTAGTTCTGTGTACTCAACGTGGAAAAGAGAAAACAGTCAGACTAAACTACAGGAGAAATATAATAGCTGGCATCACGGTGACTTCAATTATGAACGTCAGGCAACGTTGACTATCAGTTCAGCGAGAGTTAATGATTCTGGAGTGTTCATGTGTTATGCCAATAATACTTTTGGATCAGCAAATGTCACAACAACCTTGGAAGTAGTAGATAAAGGATTCATTAATATCTTCCCCATGATAAACACTACAGTATTTGTAAACGATGGAGAAAATGTAGATTTGATTGTTGAATATGAAGCATTCCCCAAACCTGAACACCAGCAGTGGATCTATATGAACAGAACCTTCACTGATAAATGGGAAGATTATCCCAAGTCTGAGAATGAAAGTAATATCAGATACGTAAGTGAACTTCATCTAACGAGATTAAAAGGCACCGAAGGAGGCACTTACACATTCCTAGTGTCCAATTCTGACGTCAATGCTGCCATAGCATTTAATGTTTATGTGAATACAAAACCAGAAATCCTGACTTACGACAGGCTCGTGAATGGCATGCTCCAATGTGTGGCAGCAGGATTCCCAGAGCCCACAATAGATTGGTATTTTTGTCCAGGAACTGAGCAGAGATGCTCTGCTTCTGTACTGCCAGTGGATGTGCAGACACTAAACTCATCTGGGCCACCGTTTGGAAAGCTAGTGGTTCAGAGTTCTATAGATTCTAGTGCATTCAAGCACAATGGCACGGTTGAATGTAAGGCTTACAACGATGTGGGCAAGACTTCTGCCTATTTTAACTTTGCATTTAAAGGTAACAACAAAGAGCAAATCCATCCCCACACCCTGTTCACTCCTTTGCTGATTGGTTTCGTAATCGTAGCTGGCATGATGTGCATTATTGTGATGATTCTGACCTACAAATATTTACAGAAACCCATGTATGAAGTACAGTGGAAGGTTGTTGAGGAGATAAATGGAAACAATTATGTTTACATAGACCCAACACAACTTCCTTATGATCACAAATGGGAGTTTCCCAGAAACAGGCTGAGTTTTGGGAAAACCCTGGGTGCTGGAGCTTTCGGGAAGGTTGTTGAGGCAACTGCTTATGGCTTAATTAAGTCAGATGCGGCCATGACTGTCGCTGTAAAGATGCTCAAGCCGAGTGCCCATTTGACAGAACGGGAAGCCCTCATGTCTGAACTCAAAGTCCTGAGTTACCTTGGTAATCACATGAATATTGTGAATCTACTTGGAGCCTGCACCATTGGAGGGCCCACCCTGGTCATTACAGAATATTGTTGCTATGGTGATCTTTTGAATTTTTTGAGAAGAAAACGTGATTCATTTATTTGTTCAAAGCAGGAAGATCATGCAGAAGCTGCACTTTATAAGAATCTTCTGCATTCAAAGGAGTCTTCCTGCAGCGATAGTACTAATGAGTACATGGACATGAAACCTGGAGTTTCTTATGTTGTCCCAACCAAGGCCGACAAAAGGAGATCTGTGAGAATAGGCTCATACATAGAAAGAGATGTGACTCCCGCCATCATGGAGGATGACGAGTTGGCCCTAGACTTAGAAGACTTGCTGAGCTTTTCTTACCAGGTGGCAAAGGGCATGGCTTTCCTCGCCTCCAAGAATTGTATTCACAGAGACTTGGCAGCCAGAAATATCCTCCTTACTCATGGTCGGATCACAAAGATTTGTGATTTTGGTCTAGCCAGAGACATCAAGAATGATTCTAATTATGTGGTTAAAGGAAACGCTCGACTACCTGTGAAGTGGATGGCACCTGAAAGCATTTTCAACTGTGTATACACGTTTGAAAGTGACGTCTGGTCCTATGGGATTTTTCTTTGGGAGCTGTTCTCTTTAGGAAGCAGCCCCTATCCTGGAATGCCGGTCGATTCTAAGTTCTACAAGATGATCAAGGAAGGCTTCCGGATGCTCAGCCCTGAACACGCACCTGCTGAAATGTATGACATAATGAAGACTTGCTGGGATGCAGATCCCCTAAAAAGACCAACATTCAAGCAAATTGTTCAGCTAATTGAGAAGCAGATTTCAGAGAGCACCAATCATATTTACTCCAACTTAGCAAACTGCAGCCCCAACCGACAGAAGCCCGTGGTAGACCATTCTGTGCGGATCAATTCTGTCGGCAGCACCGCTTCCTCCTCCCAGCCTCTGCTTGTGCACGACGATGTCTGAGCAGAATCAGTGTTTGGGTCACCCCTCCAGGAATGATCTCTTCTTTTGGCTTCCATGATGGTTATTTTCTTTTCTTTCAACTTGCATCCAACTCCAGGATAGTGGGCACCCCACTGCAATCCTGTCTTTCTGAGCACACTTTAGTGGCCGATGATTTTTGTCATCAGCCACCATCCTATTGCAAAGGTTCCAACTGTATATATTCCCAATAGCAACGTAGCTTCTACCATGAACAGAAAACATTCTGATTTGGAAAAAGAGAGGGAGGTATGGACTGGGGGCCAGAGTCCTTTCCAAGGCTTCTCCAATTCTGCCCAAAAATATGGTTGATAGTTTACCTGAATAAATGGTAGTAATCACAGTTGGCCTTCAGAACCATCCATAGTAGTATGATGATACAAGATTAGAAGCTGAAAACCTAAGTCCTTTATGTGGAAAACAGAACATCATTAGAACAAAGGACAGAGTATGAACACCTGGGCTTAAGAAATCTAGTATTTCATGCTGGGAATGAGACATAGGCCATGAAAAAAATGATCCCCAAGTGTGAACAAAAGATGCTCTTCTGTGGACCACTGCATGAGCTTTTATACTACCGACCTGGTTTTTAAATAGAGTTTGCTATTAGAGCATTGAATTGGAGAGAAGGCCTCCCTAGCCAGCACTTGTATATACGCATCTATAAATTGTCCGTGTTCATACATTTGAGGGGAAAACACCATAAGGTTTCGTTTCTGTATACAACCCTGGCATTATGTCCACTGTGTATAGAAGTAGATTAAGAGCCATATAAGTTTGAAGGAAACAGTTAATACCATTTTTTAAGGAAACAATATAACCACAAAGCACAGTTTGAACAAAATCTCCTCTTTTAGCTGATGAACTTATTCTGTAGATTCTGTGGAACAAGCCTATCAGCTTCAGAATGGCATTGTACTCAATGGATTTGATGCTGTTTGACAAAGTTACTGATTCACTGCATGGCTCCCACAGGAGTGGGAAAACACTGCCATCTTAGTTTGGATTCTTATGTAGCAGGAAATAAAGTATAGGTTTAGCCTCCTTCGCAGGCATGTCCTGGACACCGGGCCAGTATCTATATATGTGTATGTACGTTTGTATGTGTGTAGACAAATATTTGGAGGGGTATTTTTGCCCTGAGTCCAAGAGGGTCCTTTAGTACCTGAAAAGTAACTTGGCTTTCATTATTAGTACTGCTCTTGTTTCTTTTCACATAGCTGTCTAGAGTAGCTTACCAGAAGCTTCCATAGTGGTGCAGAGGAAGTGGAAGGCATCAGTCCCTATGTATTTGCAGTTCACCTGCACTTAAGGCACTCTGTTATTTAGACTCATCTTACTGTACCTGTTCCTTAGACCTTCCATAATGCTACTGTCTCACTGAAACATTTAAATTTTACCCTTTAGACTGTAGCCTGGATATTATTCTTGTAGTTTACCTCTTTAAAAACAAAACAAAACAAAACAAAAAACTCCCCTTCCTCACTGCCCAATATAAAAGGCAAATGTGTACATGGCAGAGTTTGTGTGTTGTCTTGAAAGATTCAGGTATGTTGCCTTTATGGTTTCCCCCTTCTACATTTCTTAGACTACATTTAGAGAACTGTGGCCGTTATCTGGAAGTAACCATTTGCACTGGAGTTCTATGCTCTCGCACCTTTCCAAAGTTAACAGATTTTGGGGTTGTGTTGTCACCCAAGAGATTGTTGTTTGCCATACTTTGTCTGAAAAATTCCTTTGTGTTTCTATTGACTTCAATGATAGTAAGAAAAGTGGTTGTTAGTTATAGATGTCTAGGTACTTCAGGGGCACTTCATTGAGAGTTTTGTCTTGGATATTCTTGAAAGTTTATATTTTTATAATTTTTTCTTACATCAGATGTTTCTTTGCAGTGGCTTAATGTTTGAAATTATTTTGTGGCTTTTTTTGTAAATATTGAAATGTAGCAATAATGTCTTTTGAATATTCCCAAGCCCATGAGTCCTTGAAAATATTTTTTATATATACAGTAACTTTATGTGTAAATACATAAGCGGCGTAAGTTTAAAGGATGTTGGTGTTCCACGTGTTTTATTCCTGTATGTTGTCCAATTGTTGACAGTTCTGAAGAATTCTAATAAAATGTACATATATAAATCAAAAAAAAAAAAAAAA (SEQ ID NO: 209) Human C-KIT protein, transcript variant 2TCTGGGGGCTCGGCTTTGCCGCGCTCGCTGCACTTGGGCGAGAGCTGGAACGTGGACCAGAGCTCGGATCCCATCGCAGCTACCGCGATGAGAGGCGCTCGCGGCGCCTGGGATTTTCTCTGCGTTCTGCTCCTACTGCTTCGCGTCCAGACAGGCTCTTCTCAACCATCTGTGAGTCCAGGGGAACCGTCTCCACCATCCATCCATCCAGGAAAATCAGACTTAATAGTCCGCGTGGGCGACGAGATTAGGCTGTTATGCACTGATCCGGGCTTTGTCAAATGGACTTTTGAGATCCTGGATGAAACGAATGAGAATAAGCAGAATGAATGGATCACGGAAAAGGCAGAAGCCACCAACACCGGCAAATACACGTGCACCAACAAACACGGCTTAAGCAATTCCATTTATGTGTTTGTTAGAGATCCTGCCAAGCTTTTCCTTGTTGACCGCTCCTTGTATGGGAAAGAAGACAACGACACGCTGGTCCGCTGTCCTCTCACAGACCCAGAAGTGACCAATTATTCCCTCAAGGGGTGCCAGGGGAAGCCTCTTCCCAAGGACTTGAGGTTTATTCCTGACCCCAAGGCGGGCATCATGATCAAAAGTGTGAAACGCGCCTACCATCGGCTCTGTCTGCATTGTTCTGTGGACCAGGAGGGCAAGTCAGTGCTGTCGGAAAAATTCATCCTGAAAGTGAGGCCAGCCTTCAAAGCTGTGCCTGTTGTGTCTGTGTCCAAAGCAAGCTATCTTCTTAGGGAAGGGGAAGAATTCACAGTGACGTGCACAATAAAAGATGTGTCTAGTTCTGTGTACTCAACGTGGAAAAGAGAAAACAGTCAGACTAAACTACAGGAGAAATATAATAGCTGGCATCACGGTGACTTCAATTATGAACGTCAGGCAACGTTGACTATCAGTTCAGCGAGAGTTAATGATTCTGGAGTGTTCATGTGTTATGCCAATAATACTTTTGGATCAGCAAATGTCACAACAACCTTGGAAGTAGTAGATAAAGGATTCATTAATATCTTCCCCATGATAAACACTACAGTATTTGTAAACGATGGAGAAAATGTAGATTTGATTGTTGAATATGAAGCATTCCCCAAACCTGAACACCAGCAGTGGATCTATATGAACAGAACCTTCACTGATAAATGGGAAGATTATCCCAAGTCTGAGAATGAAAGTAATATCAGATACGTAAGTGAACTTCATCTAACGAGATTAAAAGGCACCGAAGGAGGCACTTACACATTCCTAGTGTCCAATTCTGACGTCAATGCTGCCATAGCATTTAATGTTTATGTGAATACAAAACCAGAAATCCTGACTTACGACAGGCTCGTGAATGGCATGCTCCAATGTGTGGCAGCAGGATTCCCAGAGCCCACAATAGATTGGTATTTTTGTCCAGGAACTGAGCAGAGATGCTCTGCTTCTGTACTGCCAGTGGATGTGCAGACACTAAACTCATCTGGGCCACCGTTTGGAAAGCTAGTGGTTCAGAGTTCTATAGATTCTAGTGCATTCAAGCACAATGGCACGGTTGAATGTAAGGCTTACAACGATGTGGGCAAGACTTCTGCCTATTTTAACTTTGCATTTAAAGAGCAAATCCATCCCCACACCCTGTTCACTCCTTTGCTGATTGGTTTCGTAATCGTAGCTGGCATGATGTGCATTATTGTGATGATTCTGACCTACAAATATTTACAGAAACCCATGTATGAAGTACAGTGGAAGGTTGTTGAGGAGATAAATGGAAACAATTATGTTTACATAGACCCAACACAACTTCCTTATGATCACAAATGGGAGTTTCCCAGAAACAGGCTGAGTTTTGGGAAAACCCTGGGTGCTGGAGCTTTCGGGAAGGTTGTTGAGGCAACTGCTTATGGCTTAATTAAGTCAGATGCGGCCATGACTGTCGCTGTAAAGATGCTCAAGCCGAGTGCCCATTTGACAGAACGGGAAGCCCTCATGTCTGAACTCAAAGTCCTGAGTTACCTTGGTAATCACATGAATATTGTGAATCTACTTGGAGCCTGCACCATTGGAGGGCCCACCCTGGTCATTACAGAATATTGTTGCTATGGTGATCTTTTGAATTTTTTGAGAAGAAAACGTGATTCATTTATTTGTTCAAAGCAGGAAGATCATGCAGAAGCTGCACTTTATAAGAATCTTCTGCATTCAAAGGAGTCTTCCTGCAGCGATAGTACTAATGAGTACATGGACATGAAACCTGGAGTTTCTTATGTTGTCCCAACCAAGGCCGACAAAAGGAGATCTGTGAGAATAGGCTCATACATAGAAAGAGATGTGACTCCCGCCATCATGGAGGATGACGAGTTGGCCCTAGACTTAGAAGACTTGCTGAGCTTTTCTTACCAGGTGGCAAAGGGCATGGCTTTCCTCGCCTCCAAGAATTGTATTCACAGAGACTTGGCAGCCAGAAATATCCTCCTTACTCATGGTCGGATCACAAAGATTTGTGATTTTGGTCTAGCCAGAGACATCAAGAATGATTCTAATTATGTGGTTAAAGGAAACGCTCGACTACCTGTGAAGTGGATGGCACCTGAAAGCATTTTCAACTGTGTATACACGTTTGAAAGTGACGTCTGGTCCTATGGGATTTTTCTTTGGGAGCTGTTCTCTTTAGGAAGCAGCCCCTATCCTGGAATGCCGGTCGATTCTAAGTTCTACAAGATGATCAAGGAAGGCTTCCGGATGCTCAGCCCTGAACACGCACCTGCTGAAATGTATGACATAATGAAGACTTGCTGGGATGCAGATCCCCTAAAAAGACCAACATTCAAGCAAATTGTTCAGCTAATTGAGAAGCAGATTTCAGAGAGCACCAATCATATTTACTCCAACTTAGCAAACTGCAGCCCCAACCGACAGAAGCCCGTGGTAGACCATTCTGTGCGGATCAATTCTGTCGGCAGCACCGCTTCCTCCTCCCAGCCTCTGCTTGTGCACGACGATGTCTGAGCAGAATCAGTGTTTGGGTCACCCCTCCAGGAATGATCTCTTCTTTTGGCTTCCATGATGGTTATTTTCTTTTCTTTCAACTTGCATCCAACTCCAGGATAGTGGGCACCCCACTGCAATCCTGTCTTTCTGAGCACACTTTAGTGGCCGATGATTTTTGTCATCAGCCACCATCCTATTGCAAAGGTTCCAACTGTATATATTCCCAATAGCAACGTAGCTTCTACCATGAACAGAAAACATTCTGATTTGGAAAAAGAGAGGGAGGTATGGACTGGGGGCCAGAGTCCTTTCCAAGGCTTCTCCAATTCTGCCCAAAAATATGGTTGATAGTTTACCTGAATAAATGGTAGTAATCACAGTTGGCCTTCAGAACCATCCATAGTAGTATGATGATACAAGATTAGAAGCTGAAAACCTAAGTCCTTTATGTGGAAAACAGAACATCATTAGAACAAAGGACAGAGTATGAACACCTGGGCTTAAGAAATCTAGTATTTCATGCTGGGAATGAGACATAGGCCATGAAAAAAATGATCCCCAAGTGTGAACAAAAGATGCTCTTCTGTGGACCACTGCATGAGCTTTTATACTACCGACCTGGTTTTTAAATAGAGTTTGCTATTAGAGCATTGAATTGGAGAGAAGGCCTCCCTAGCCAGCACTTGTATATACGCATCTATAAATTGTCCGTGTTCATACATTTGAGGGGAAAACACCATAAGGTTTCGTTTCTGTATACAACCCTGGCATTATGTCCACTGTGTATAGAAGTAGATTAAGAGCCATATAAGTTTGAAGGAAACAGTTAATACCATTTTTTAAGGAAACAATATAACCACAAAGCACAGTTTGAACAAAATCTCCTCTTTTAGCTGATGAACTTATTCTGTAGATTCTGTGGAACAAGCCTATCAGCTTCAGAATGGCATTGTACTCAATGGATTTGATGCTGTTTGACAAAGTTACTGATTCACTGCATGGCTCCCACAGGAGTGGGAAAACACTGCCATCTTAGTTTGGATTCTTATGTAGCAGGAAATAAAGTATAGGTTTAGCCTCCTTCGCAGGCATGTCCTGGACACCGGGCCAGTATCTATATATGTGTATGTACGTTTGTATGTGTGTAGACAAATATTTGGAGGGGTATTTTTGCCCTGAGTCCAAGAGGGTCCTTTAGTACCTGAAAAGTAACTTGGCTTTCATTATTAGTACTGCTCTTGTTTCTTTTCACATAGCTGTCTAGAGTAGCTTACCAGAAGCTTCCATAGTGGTGCAGAGGAAGTGGAAGGCATCAGTCCCTATGTATTTGCAGTTCACCTGCACTTAAGGCACTCTGTTATTTAGACTCATCTTACTGTACCTGTTCCTTAGACCTTCCATAATGCTACTGTCTCACTGAAACATTTAAATTTTACCCTTTAGACTGTAGCCTGGATATTATTCTTGTAGTTTACCTCTTTAAAAACAAAACAAAACAAAACAAAAAACTCCCCTTCCTCACTGCCCAATATAAAAGGCAAATGTGTACATGGCAGAGTTTGTGTGTTGTCTTGAAAGATTCAGGTATGTTGCCTTTATGGTTTCCCCCTTCTACATTTCTTAGACTACATTTAGAGAACTGTGGCCGTTATCTGGAAGTAACCATTTGCACTGGAGTTCTATGCTCTCGCACCTTTCCAAAGTTAACAGATTTTGGGGTTGTGTTGTCACCCAAGAGATTGTTGTTTGCCATACTTTGTCTGAAAAATTCCTTTGTGTTTCTATTGACTTCAATGATAGTAAGAAAAGTGGTTGTTAGTTATAGATGTCTAGGTACTTCAGGGGCACTTCATTGAGAGTTTTGTCTTGGATATTCTTGAAAGTTTATATTTTTATAATTTTTTCTTACATCAGATGTTTCTTTGCAGTGGCTTAATGTTTGAAATTATTTTGTGGCTTTTTTTGTAAATATTGAAATGTAGCAATAATGTCTTTTGAATATTCCCAAGCCCATGAGTCCTTGAAAATATTTTTTATATATACAGTAACTTTATGTGTAAATACATAAGCGGCGTAAGTTTAAAGGATGTTGGTGTTCCACGTGTTTTATTCCTGTATGTTGTCCAATTGTTGACAGTTCTGAAGAATTCTAATAAATGTACATATATAAATCAAAAAAAAA(SEQ ID NO: 210)

The invention claimed is:
 1. A method for treating cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of an anti-C-KIT antibody or an antigen-binding portion thereof,wherein the antibody comprises a heavy chain variable (VH) region and alight chain variable (VL) region, wherein (a) the VH region amino acidsequence comprises HCDR1 of GYTFTDYYMN (SEQ ID NO: 13), HCDR2 ofMGRIYPGSGNTYYAQKFQG (SEQ ID NO: 14) and HCDR3 of GVYYYDY (SEQ ID NO:15); and the VL region amino acid sequence comprises LCDR1 ofRASQGIRTNLA (SEQ ID NO: 16), LCDR2 of AASSLQS (SEQ ID NO: 24) and LCDR3of QQYNSYPRT (SEQ ID NO: 12); or (b) the VH region amino acid sequencecomprises HCDR1 of GYTFTDYYMN (SEQ ID NO: 13), HCDR2 ofMGRIYPGSGNTYYAQKFQG (SEQ ID NO: 14) and HCDR3 of GVYYYDY (SEQ ID NO:15); and the VL region amino acid sequence comprises LCDR1 ofRASQGIRTNLA (SEQ ID NO: 16), LCDR2 of AASSLQS (SEQ ID NO: 24) and LCDR3of QQYANYPRT (SEQ ID NO: 25); and wherein the cancer is gastrointestinalstromal cancer (GIST), lung cancer, acute myeloid leukemia (AML) or amast cell tumor.
 2. The method of claim 1, wherein (a) the VH regionamino acid sequence comprises SEQ ID NO:185 and the VL region amino acidsequence comprises SEQ ID NO:186; or (b) the VH region amino acidsequence comprises SEQ ID NO:187 and the VL region amino acid sequencecomprises SEQ ID NO:188.
 3. The method of claim 1, wherein the antibodyis humanized or chimeric.
 4. The method of claim 1, wherein the VHregion, the VL region, or both the VH and the VL region comprise one ormore human framework region amino acid sequences.
 5. The method of claim1, wherein the VH region, the VL region, or both the VH and the VLregion comprise a human variable region framework scaffold amino acidsequence into which the CDRs have been inserted.
 6. The method of claim1, wherein the VH region comprises an IGHV1-46 human germline scaffoldamino acid sequence into which the HCDR1, HCDR2 and HCDR3 amino acidsequences have been inserted.
 7. The method of claim 1, wherein the VLregion comprises an IGKV1-16 human germline scaffold amino acid sequenceinto which the LCDR1, LCDR2 and LCDR3 amino acid sequences have beeninserted.
 8. The method of claim 1, wherein the antibody comprises animmunoglobulin constant region.
 9. The method of claim 8, wherein theimmunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY. 10.The method of claim 9, wherein the immunoglobulin constant region isIgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.
 11. The method of claim 8, whereinthe immunoglobulin constant region is immunologically inert.
 12. Themethod of claim 9, wherein the immunoglobulin constant region is awild-type human IgG1 constant region, a human IgG1 constant regioncomprising the amino acid substitutions L234A, L235A and G237A or ahuman IgG1 constant region comprising the amino acid substitutionsL234A, L235A, G237A and P331S.
 13. The method of claim 9, wherein theimmunoglobulin constant region comprises SEQ ID NO:197, SEQ ID NO:198,SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201 or SEQ ID NO:202.
 14. Themethod of claim 1, wherein the antibody or antigen-binding portion is anFab, an Fab′, an F(ab′)2, an Fv, an scFv, a maxibody, a minibody, adiabody, a triabody, a tetrabody, or a bis-scFv.
 15. The method of claim1, wherein the antibody is monoclonal.
 16. The method of claim 1,wherein the antibody or antigen-binding portion binds specifically to(a) human C-KIT or (b) human C-KIT and cynomolgus C-KIT.
 17. The methodof claim 1, wherein the antibody or antigen-binding portion is linked toa therapeutic agent.
 18. The method of claim 17, wherein the therapeuticagent is a cytotoxin, a radioisotope, a chemotherapeutic agent, animmunomodulatory agent, a cytostatic enzyme, a cytolytic enzyme, atherapeutic nucleic acid, an anti-angiogenic agent, ananti-proliferative agent, or a pro-apoptotic agent.
 19. The method ofclaim 1, wherein the antibody or antigen-binding portion is administeredto the subject in the form of a pharmaceutical composition comprisingthe antibody or antigen-binding portion and a pharmaceuticallyacceptable carrier, diluent or excipient.
 20. The method of claim 1,wherein the antibody or antigen-binding portion is administered to thesubject in a combination with an additional therapeutic agent.
 21. Themethod of claim 20, wherein the additional therapeutic agent is ananti-cancer agent.